Non-myeloablative tolerogenic treatment

ABSTRACT

The present invention features a method of inducing donor-specific tolerance in a host. Tolerogenic treatments of the present invention may be administered to a host prior to transplantation of donor-derived materials. The tolerogenic treatment involves (1) administering an immunosuppressive agent to a host mammal in a non-myeloablative regimen sufficient to decrease, but not necessarily to eliminate, the host mammal&#39;s functional T lymphocyte population; (2) infusing donor antigens from a non-syngeneic donor into the host mammal; (3) eliminating those host T lymphocytes responding to the infused donor antigens using a non-myeloablative dose of lymphocytotoxic or tolerizing agent; and (4) administering donor hematopoietic cells to the host mammal. Donor lymphoid cells used for cell therapy of a host mammal can be depleted of host specific immunological reactivity by methods essentially similar to those use for tolerizing a host mammal prior to transplantation.

This application claims priority of International Application No.US98/10575, filed May 22, 1998, and is a continuation in part U.S.application Ser. No. 08/862,550, filed May 23, 1997, now abandoned.

BACKGROUND OF THE INVENTION

Transplantation of organs, hematopoietic cells and somatic cells hasbeen a crucial therapeutic regimen for patients suffering from a varietyof maladies. Although the techniques necessary for transplants are quitestraight-forward, the great stumbling block for successfultransplantation has been the immune system. A fundamental problem hasbeen the great vigor with which the host immune system reacts againstintroduction of antigens found in donor tissues or cells.

Transplantation of allogeneic donor (i.e., the same species but notgenetically identical to the host patient) or xenogeneic donor (i.e., aspecies other than that of the host) grafts has posed particularly greatdifficulties. The continued functioning of any donor graft depends uponcontinued functioning of the donor cells that make up that graft. Thecells of donor grafts, however, can elicit an immune reaction on thepart of the host that, if unchecked, may lead to destruction of thegraft.

One method of alleviating the reaction by the host against a graft hasbeen administration of immunosuppressive treatment to the host.Unfortunately, despite the availability of new and very effectiveimmunosuppressive drugs, recurrent episodes of acute and chronic graftrejection remain common, frequently causing loss of graft function.Moreover, the long-term success of transplantation is often limited bycomplications resulting from drug-related toxicity and from long-termimmunosuppression (e.g. infections and secondary malignancies). Inaddition, transplantation of bone marrow cells (BMC) or small intestine,which are rich in immunocompetent lymphocytes, frequently is associatedwith a potential life-threatening complication due to graft versus hostdisease (GVHD).

It has been shown that a full hematopoietic chimera, i.e., a patientwhose own BMC have been 100% replaced by permanently engrafted BMC fromanother individual (donor), can permanently accept donor-derivedallografts with no need for maintenance immunosuppressive therapy.However, induction of full hematopoietic chimerism has been difficult toaccomplish. First, substantially complete destruction of the host'simmunohematopoietic compartment (“lethal” conditioning) is usuallyrequired for engraftment of matched and especially mismatched BMC. Withlethal conditioning of the host, GVHD consistently causes morbidity ormortality. In such cases, T cell depletion of the graft hematopoieticmaterial represents the only approach for effective prevention of GVHD.T cell depletion in turn is associated with an increased incidence ofgraft rejection. To overcome the problem of graft rejection, recipientsof T cell depleted marrow allografts may require particularly strongconditioning or, alternatively, very high numbers of T cell depletedBMC. Subjecting patients to aggressive rejection-prevention protocols,such as total body irradiation (TBI) alone or TBI in combination with ashort course of immunosuppressive drugs is unlikely to be accepted byclinicians treating patients in need of organ allografts.

It has been proposed that true bilateral tolerance associated with mixeddonor/recipient hematopoietic chimerism, i.e., the condition in which apatient possesses both recipient (host) and donor hematopoietic stemcells, rather than with full chimerism, would be preferable in clinicalorgan transplantation. Several experimental protocols have been designedto induce transplantation tolerance leading to mixed chimerism.Conditioning has required the use of high dose TBI followed by infusionwith a mixture of T cell depleted donor and recipient BMC (Sachs et al.,Ann. Thorac. Surg., 56:1221 (1993); Ildstad et al., Nature, 307:168(1984)) or inoculation with donor BMC after lower dose TBI and infusionof a mixture of antibodies against CD4⁺ T cells, CD8⁺ T cells and NKcells leading to general pancytopenia. Tomita et al., J. Immunol.,153:1087 (1994); Tomita et al., Transplantation, 61:469 (1996). Analternative approach has been developed recently involving irradiationwith a sublethal dose of TBI and inoculation with a very high number ofT cell depleted donor-derived hematopoietic cells. Reisner et al.,Immunol. Today, 16:437 (1995); Bachar-Lustig et al., Nature Medicine,12:1268 (1986). Tolerogenic treatments using cyclophosphamide(hereinafter also referred to as “Cytoxan” or “Cy”) in combination withTBI have also been described.

Total lymphoid irradiation (TLI) has been employed successfully as thesole preparatory regimen prior to infusion with donor BMC, to inducemixed hematopoietic chimerism and bilateral transplantation tolerance.Slavin S., Immunol. Today, 3:88 (1987); Slavin et al., Isr. J. Med.Sci., 22:264 (1986). TLI is non-myeloablative and routinely given safelyon an outpatient basis to transplant recipients and patients withHodgkin's disease. Unfortunately, consistent induction of chimerismusing TLI has required very high cumulative doses of radiation(3,400-4,400 cGy) that again would not be desirable for transplantrecipients. TLI has significant advantages over TBI, especially in theclinical setting. TLI, which involves selective irradiation of thelymphoid compartment without exposing the whole body to ionizingirradiation, is well tolerated. In addition, TLI preserves intact asignificant portion of the host's immunohematopoietic system, withresultant retained memory to recall antigens including infective agents.However, long courses of TLI can be time consuming and may be associatedwith short and long-term side effects that may not be suitable forroutine clinical application.

SUMMARY OF THE INVENTION

The invention provides a new method for treating a host mammal to inducetransplantation tolerance to cell, tissue and organ allografts andxenografts. Such transplants can provide replacement therapy for enzymeor metabolic disorders and adoptive immunotherapy for cancer andlife-threatening infections in humans. The method also can be used toprovide new animal models for tolerance induction toward allogeneic andxenogeneic cells. The invention also provides a new method ofnon-syngeneic cell therapy in which the cell population used for therapyis substantially depleted of responsiveness to host antigens prior toadministration to the host.

In general, the invention features a method of treating a host mammal,including (a) administering donor antigens from a non-syngeneic donor tothe host mammal; (b) administering a non-myeloablative dose oflymphocytotoxic agent (e.g., cyclophosphamide) or tolerizing agent tothe host mammal to selectively eliminate the host mammal's lymphocytesresponding to the donor antigens; and (c) administering a preparation ofhematopoietic stem cells from the non-syngeneic donor to the hostmammal.

Prior to step (a), the host mammal can be administered animmunosuppressive agent in a non-myeloablative regimen sufficient todecrease the host mammal's functional T lymphocyte population. Theimmunosuppressive agent can include one or more of an immunosuppressivedrug, an alkylating agent, ionizing radiation, or anti-leukocyte oranti-leukocyte function antibodies. It is particularly advantageous touse a short course of TLI (sTLI) as the immunosuppressive agent, forexample 1-12, frequently 1-6, doses of 200 cGy/dose.

The donor antigens administered to the host mammal can includenon-cellular antigens, cells, tissues and/or organs. For example, thedonor antigens can include hematopoietic stem cells or other viablecells. If the donor antigens include viable cells such as hematopoieticstem cells, then the immunosuppressive regimen referenced above shoulddecrease the T lymphocyte population of the host to a level permittingat least transient survival of the donor's cells. For example the Tlymphocyte population of the host can be decreased by 90%, 95% or 99%

The host mammal can be an animal or a human, for example a human cancerpatient. The donor can be allogeneic or xenogeneic to the host mammal.Following performance of the method, the host mammal's blood can contain20% or more donor cells. After administering the preparation of donorhematopoietic stem cells, with resultant engraftment of such cells inthe host, the host can be treated with allogeneic cell therapy. Thisinvolves infusing allogeneic lymphocytes from the donor into the hostmammal. Alternatively, the host can receive transplanted cells, tissuesor organs from the donor, with the transplants becoming engrafted in thehost due to the donor-specific tolerance induced in the host mammal.

In another aspect, the invention features a host-derived hematopoieticcell composition, including host-originating and donor-originatinghematopoietic cells, with the composition being depleted ofdonor-specific, host-originating lymphocytes. The hematopoietic cellcomposition can be made by treating a host mammal as described above,then isolating the hematopoietic cell composition from the host mammal.

In a further aspect, the invention features a method of making anon-human mammal/human chimera. This involves performing the methodsdescribed above, with the host mammal being a non-human mammal and thedonor being a human being. The host mammal can be, for example, a rodentor pig. The result is a rodent, pig or other non-human mammal stablyengrafted with human hematopoietic stem cells. As such, the non-humanmammal host constitutes a hematopoietic mixed chimera.

The invention also encompasses a composition of cells containing a cellpopulation from a first individual mammal. The cell population containslymphocytes and is depleted of responsiveness to antigens of a secondindividual mammal that is non-syngeneic (i.e., allogeneic or xenogeneic)with the first individual mammal. The depletion of responsiveness is bya method involving the following sequential steps: (a) administering anantigen source expressed by the second individual mammal to the firstindividual mammal; (b) administering a non-myeloablative dose of alymphocytotoxic or tolerizing agent to the first individual mammal; (c)administering a preparation of hemopoietic cells from the secondindividual mammal to the first individual mammal; and (d) isolating thecell population from the first individual mammal. In the composition ofthe invention, cells endogenous to the first individual mammal are 50%to 100% of the cells of the population. The antigen used can be cancercells and the first individual mammal and the second individual mammalcan both be humans. Alternatively, the first individual mammal can be anon-human primate, and said second individual mammal can be a human, orthe first individual mammal can be a pig and said second individualmammal can a human.

The invention also features a method of treating a mammal withnon-syngeneic cell therapy. The method involves infusing a population ofcells from a donor mammal into a host mammal, with the donor mammal andthe host mammal being non-syngeneic with each other. The cell populationcan contain lymphocytes, and prior to infusing, the cell population canbe depleted of responsiveness to antigens expressed by the host mammal.The depletion of responsiveness can be by substantially eliminating Tcells from the cell population. Elimination of T cells can be byexposing the cell population to an immunosuppressive agent in anon-myeloablative regimen or by contacting the cell with mafosphamide.These eliminations can be performed in vitro or in vivo.

Alternatively, the depletion of the cell population can be accomplishedby contacting the lymphocyte population with a composition comprisingantigens expressed by said host mammal and the contacting can be invitro or by administering the antigens to the first individual mammal.The method can further include the step of, after the contacting withthe antigen composition, delivering a non-myeloablative dose of alymphocytotoxic or tolerizing agent to the lymphocyte population. Thisdelivering can be in vitro or by administering the non-myeloablativedose to the donor mammal. The method can also optionally include thesteps of: (a) after the delivering, administering a preparation ofhemopoietic stem cells from the host mammal to the donor mammal; and/or(b) prior to the contacting with antigen, exposing the lymphocytepopulation to an immunosuppressive agent in a non-myeloablative regimensufficient to decrease the number of functional T lymphocytes in thelymphocyte population. In (b) the exposing can be in vitro or byadministering the immunosuppressive agent to the donor mammal. Theantigen composition can contain one or more antigen sources, e.g.,cells, organs, tissues, and non-cellular antigens. For example, theantigen can include hemopoietic cells or cancer cells expressing majorhistocompatibility complex molecules of the host mammal. The cancercells can, for example, be from the host mammal.

Also within the invention is an article of manufacture that includespackaging material and a biological cell container within the packagingmaterial. The cell container can contain a composition that includeshematopoietic stem cells and the packaging material can contain a labelor package insert indicating that the hematopoietic stem cells are to beused in step (a) or step (c) in a method of inducing non-syngeneicdonor-specific tolerance in a host mammal. The method includes the stepsof: (a) administering donor antigens from a non-syngeneic donor to thehost mammal; (b) administering a non-myeloablative dose oflymphocytotoxic or tolerizing agent to the host mammal to selectivelyeliminate the host mammal's lymphocytes responding to the donorantigens; and (c) administering a preparation of hematopoietic stemcells from the non-syngeneic donor to the host mammal.

Another article of manufacture encompassed by the invention is one thatincludes packaging material, a biological cell container within saidpackaging material, with the cell container containing any of the cellcompositions of the invention described above. The packaging materialcontains a label or package insert indicating that the composition is tobe used in a method of treatment including administering the compositionto a second individual mammal that is in need of the composition.

The invention also features a method of inducing tolerance in a hostmammal to a graft from a non-syngeneic host mammal. The method includesthe following steps: (a) administering donor antigens from anon-syngeneic donor to the host mammal; (b) administering animmunosuppressive agent to the host mammal in a non-myeloablativeregimen sufficient to decrease the host mammal's functional T lymphocytepopulation; (c) transplanting cells, a tissue, or an organ from thedonor into the host animal, (d) administering a non-myeloablative doseof lymphocytotoxic or tolerizing agent to the host mammal to selectivelyeliminate the host mammal's lymphocytes responding to the donorantigens; and (e) administering a preparation of hematopoietic stemcells from the non-syngeneic donor to the host mammal. Steps (a), (b),and (c) of the method are performed on the same day and prior to steps(d) and (e).

The term “non-myeloablative” as used herein includes any therapy thatdoes not eliminate substantially all hematopoietic cells of host origin.“Transplantation” as used herein refers to transplantation of anydonor-derived material including cells, tissues and organs. The cellsmay be hematopoietic or non-hematopoietic. “Donor antigens” as usedherein refers to any donor-derived material that elicits a host immuneresponse, including non-cellular antigens, cells, tissues or organs.Stem cells are particularly useful as donor antigens. A “lymphocytotoxicagent” is an agent that kills T cells or paralyzes T cell function. A“tolerizing agent” is an agent that energizes or “vetos” T cells bypreventing development of normal T cell-dependent responses. The term“cancer” as used herein includes all pathological conditions involvingmalignant cells; this can include “solid” tumors arising in solidtissues or organs as well as hematopoietic tumors such as leukemias andlymphomas. The term “donor-specific tolerance” as used herein refers totolerance of the host to donor-derived material. “Non-syngeneic” as usedherein can be allogeneic or xenogeneic. “Depletion of responsiveness” ina particular cell population, as used herein, means either a decrease inthe number of responsive cells, a decrease in the responsiveness ofresponsive cells, or both. Where cells are herein said to be“endogenous” to an individual mammal, it is understood that the cellsthemselves, or their precursors, were in that individual mammal prior toany administration of cells from another individual mammal.

Induction of donor-specific tolerance across strong majorhistocompatibility complex MHC and minor histocompatibility loci (MiHL)barriers, as well as across species barriers (xenogeneic tolerance) maybe achieved in mammalian hosts using the tolerogenic treatment describedherein. Induction of donor-specific transplantation tolerance whileavoiding the need for maintenance immunosuppressive treatment is ahighly desirable goal in clinical transplantation.

The non-myeloablative tolerogenic treatment described herein induces astate of long-lasting donor-specific tolerance to a wide variety ofdonor-derived material. Such an approach is attractive for allogeneicand xenogeneic transplantation of cells, tissues and organs in clinicalsettings, since all the steps of the protocol are well tolerated andrelatively safe. Since there is no need to eradicate the entire hostimmunohematopoietic system during the course of the procedure, therecipients retain immune memory and are in a better position to resistgraft-versus-host disease on the one hand and infectious complicationson the other. This can be of crucial importance in clinical practice.The protocols for inducing donor-specific tolerance may be delivered, atleast in part, as outpatient procedures.

The methods of non-syngeneic cell therapy provided herein can beespecially useful in conditions in which cell, tissue, or organ failureor misfunction occurs. They can therefore be useful in, for example,metabolic deficiencies (including genetic metabolic deficiencies),autoimmune diseases, and cancer. The methods are therefore useful inpassively transferring, from a donor to a host, immunity to one moreinfectious agents. They can be used without prior treatment of the hostor subsequent to tolerization of the host to donor antigens by one ofthe tolerization methods of the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of T cell depletion from a second BMC infusion onsurvival of tolerant mice.

FIGS. 2.(A, B, & C) GVHD-free survival (upper panel) and donor-type skinallograft survival (lower panel) of BALB/c mice irradiated with variousnumber of daily TLI fractions.

FIG. 3. Xenograft survival of Lewis rat skin in B6 mouse recipientsfollowing a non-myeloablative tolerogenic protocol based on the use ofsTLI, donor BMC and Cy.

FIG. 4. Survival of host F1 mice after injection of BCL1 tumor cells andlymphoid cells from either immunized or non-immunized B6 donor miceincompatible with the host mice at both the MHC and MiHL.

FIG. 5. Survival of secondary BALB/c host mice after transfer of spleencells from primary F1 host mice injected with BCL1 tumor cells andlymphoid cells from either immunized or non-immunized B6 donor miceincompatible with the host mice at both the MHC and MiHL.

FIG. 6. Body weight changes in F1 host mice after injection of BCL1tumor cells and lymphoid cells from either immunized or non-immunized B6donor mice incompatible with the host mice at both the MHC and MiHL.

FIG. 7. Survival of host BALB/c mice after injection of BCL1 tumor cellsand lymphoid cells from either immunized or non-immunized B10.D2 donormice incompatible with the host mice at MiHL only.

FIG. 8. Survival of secondary BALB/c host mice after transfer of spleencells from primary BALB/c host mice injected with BCL1 tumor cells andlymphoid cells from either immunized or non-immunized B10.D2 donor miceincompatible with the primary BALB/c host mice at MiHL only.

FIG. 9. Body weight changes in BALB/c host mice after injection of BCL1tumor cells and lymphoid cells from either immunized or non-immunizedB10.D2 donor mice incompatible with the host mice at MiHL only.

FIG. 10. Production of interferon-γ (IFN-γ) by spleen cells from C57BL/6mice immunized with either BCL1 tumor cells or BALB/c spleen cells andby spleen cells from unimmunized C57BL/6 mice.

FIG. 11. Production of interleukin-2 (IL-2) by spleen cells from C57BL/6mice immunized with either BCL1 tumor cells or BALB/c spleen cells andby spleen cells from unimmunized C57BL/6 mice.

FIG. 12. Production of interleukin-10 (IL-10) by spleen cells fromC57BL/6 mice immunized with either BCL1 tumor cells or BALB/c spleencells and by spleen cells from unimmunized C57BL/6 mice.

FIG. 13. Production of tumor necrosis factor-α (TNF-α) by spleen cellsfrom F1 mice injected with the three spleen cell populations representedin FIGS. 10-12.

FIG. 14. Production of IL-2 by spleen cells from F1 mice injected withthe three spleen cell populations represented in FIGS. 10-12.

FIG. 15. Production of IFN-γ by spleen cells from F1 mice injected withthe three spleen cell populations represented in FIGS. 10-12.

FIG. 16. Production of interleukin-4 (IL-4) by spleen cells from F1 miceinjected with the three spleen cell populations represented in FIGS.10-12.

FIG. 17. Survival of BALB/c recipient mice and allografts (bone marrowstroma or hearts) from B6 mice after tolerization and transplantationusing a protocol in which the sTLI, the first B6 bone marrow infusion,and allograft implantation were all performed on day 0.

FIG. 18. Lysis of murine YAC-1 tumor targets by killer cells generatedby in vitro activation with IL-2 of BALB/c bone marrow (BM) or spleen(SP) cells after either no treatment or treatment with ASTA-Z andsubsequent culture with IL-2.

FIG. 19. Lysis of murine P815 tumor target cells by killer cellsgenerated by in vitro activation with IL-2 of BALB/c bone marrow (BM) orspleen (SP) cells after either no treatment or treatment with ASTA-Z andsubsequent culture with IL-2.

FIG. 20. Survival of lethally irradiated SJL/J mice after infusion ofeither untreated or ASTA-Z treated bone marrow cells from B6 mice.Survival data obtained with control lethally irradiated, unreconstitutedSJL/J mice are also shown.

FIG. 21. Survival of sub-lethally irradiated BALB/c mice after infusionof either untreated or ASTA-Z treated bone marrow cells from B6 mice.

DETAILED DESCRIPTION

A. Tolerance Protocols

The present inventor has employed novel, non-myeloablative tolerogenicprotocols to induce stable and donor-specific tolerance to non-syngeneictransplants (i.e., transplants of cells, tissues or organs notgenetically identical to the host). A protocol for the tolerogenictreatment can be summarized as follows:

Step 1: Administer an immunosuppressive agent to a host mammal in anon-myeloablative regimen sufficient to decrease, but not eliminate, thehost mammal's functional T lymphocyte population.

Step 2: Infuse donor antigens, preferably viable hematopoietic cells,from a non-syngeneic donor into the host mammal.

Step 3: Eliminate those host T lymphocytes responding to the infuseddonor antigens using a non-myeloablative dose of lymphocytotoxic ortolerizing agent.

Step 4: Administer a preparation of donor hematopoietic stem cells tothe host mammal.

This non-myeloablative, donor-specific tolerogenic treatment results inconversion of a host to a hematopoietic mixed chimera with high levelsof donor hematopoietic cells. Typically, the mammalian hosts are humanpatients, although a recipient of the tolerogenic treatment may be anymammal. Non-syngeneic transplantation can include allogeneic as well asxenogeneic transplantation of organs, tissues or cells. Hence,hematopoietic stem cells and other donor antigens used in steps 2 and 4may be derived from allogeneic or xenogeneic sources.

Human patients for which the tolerogenic treatment is appropriateinclude without limitation those with loss of organ or tissue functionincluding loss of metabolic function such as in diabetes; patients withenzyme deficiencies caused by inborn genetic diseases such as Gaucher'sdisease, metachromatic leukodystrophy and Hurler's Syndrome; patientswith autoimmune disorders such as lupus erythematosus and rheumatoidarthritis; and cancer patients. Patients suffering from heart, liver orkidney failure, for example, are excellent candidates for conditioningwith the tolerogenic treatment prior to transplantation with theappropriate organ. Patients requiring a skin or bone graft may also besubjected to the tolerogenic treatment prior to grafting. Cancerpatients receiving the tolerogenic treatment can include patientssuffering from any malignancy, either solid tumors such as breast canceror hematopoietic malignancies including acute and chronic leukemia,lymphoma, and myelodysplastic and myeloproliferative disorders.

In accordance with this invention, a significant number of the hostmammal's functional T lymphocyte population remains in the host afterthe non-myeloablative regimen of Step 1. Nevertheless, engraftment ofdonor cells can occur because (a) donor-reactive host T lymphocytes areeliminated in step 3, and (b) donor-derived T lymphocytes and/or stemcells present in the subsequent infusion or infusions (Step 4) may actas “veto” cells to produce a veto effect. Veto cells, as used herein,include T lymphocytes, especially CD8⁺ T cells, that result in downregulation, rather than stimulation, of other T lymphocytes. Vetoeffects may be induced by other proliferating hematopoietic cellsincluding T cell-depleted stem cells that are poorly immunogenic butthat can veto host T cells. In the veto effect, host-originating Tlymphocytes are down-regulated by donor-derived veto cells, includingstem cells and/or lymphocytes. Other replicating donor-derived cells, oreven non-cellular antigens, can also veto host allo- or xeno-reactive Tcells if provided repeatedly and in relatively high concentrations.Similarly, immunocompetent T cells present in the donor infusion may bedown-regulated by veto cells of host origin. Thus, tolerance of graft vshost and host vs graft may occur simultaneously due to a balancedequilibrium between veto cells of host and donor origin on the one handand the degree of immunogenicity and alloreactivity of the graft on theother.

(i) Step 1

Examples of immunosuppressive agents useful in Step 1 include withoutlimitation immunosuppressive drugs such as methotrexate and fludarabine(FLU); alkylating agents such as Cy, melphalan, thiotepa and busulfan;polyclonal and monoclonal anti-thymocyte globulin (ATG) andanti-lymphocyte globulin (ALG); and ionizing radiation such as TLI andTBI. Due to its non-selective effects on all of the host's hematopoieticcells and its severe immediate and long-term side effects, TBI is notpreferred. If TBI is used, it should be at a dose level that causes nosevere or irreversible pancytopenia. The non-myeloablative regimenadvantageously is a short and well-tolerated course of TLI (sTLI) whichmay cause a major reduction in the number and/or function of host Tlymphocytes in all lymphoid organs. As discussed below, it has beendiscovered that sTLI can effectively induce unresponsiveness to donorantigens at relatively low cumulative radiation doses.

The sTLI immunosuppressive regimen may comprise, for example, 1 to 12daily fractions of 200 cGy/each depending on the host-versus-graftpotential and the T lymphocyte content in the stem cell preparationadministered in Step 4. Stem cell preparations rich in T lymphocytes mayrequire only 1-3 sTLI fractions, or may not require immunosuppression atall (zero sTLI fractions). Transplantation of T cell-depleted stem cellpreparations or stem cell preparations with low levels of T lymphocytes,however, may require the use of 4-12 fractions. The sTLI regimen causesonly a transient reduction in the number of host T lymphocytes and isclinically feasible on an outpatient basis. There are no anticipatedsevere side effects since a routine cumulative dose of TLI usedclinically for lymphoma patients consists of 4,400 cGy.

Preferably, the immunosuppressive agent transiently decreases the hostfunctional T lymphocyte population by at least about 90%. Morepreferably, the non-myeloablative regimen transiently decreases the hostfunctional T lymphocyte population by at least about 95%, and mostpreferably, by at least about 99%. Reductions of less than 90% of thelymphocytes are also within the scope of this invention, provided thattransient survival of donor antigens, provided in Step 2, is possible.

In some donor/recipient combinations, tolerance to donor antigens may beinducible without the necessity of performing Step 1. In this case, thepreparation of donor hematopoietic stem cells administered in Step 4must contain a sufficient number of T cells to provide a protective vetofunction against residual host T cells escaping the effects of Step 3.

(ii) Step 2

In Step 2 of the tolerogenic treatment, antigens from a non-syngeneicdonor are administered to the host mammal in order to stimulate andcause proliferation of donor-specific T lymphocytes of the host. Thestimulated sub-population of donor-specific host T lymphocytes is theneliminated or tolerized in Step 3. The donor antigens may beadministered (Step 2) to the host after the non-myeloablativeimmunosuppressive regimen (Step 1) described above. Alternatively, thedonor antigens may be administered to a non-immunosuppressed host (ifStep 1 is excluded as described above).

The donor antigens administered in step 2 can include, withoutlimitation, non-cellular antigens, cells, organs, tissues or tissueextracts, or even anti-idiotypic antibodies that mimic donor antigens.In general, any donor antigens that elicit an immune response in thehost are within the scope of this invention. Any source of donorantigens from a non-syngeneic donor can be used, and the non-syngeneicdonor can be allogeneic or xenogeneic to the host.

The infusion of donor antigens should comprise donor antigenicdeterminants for which tolerance is desired. For example, if it isdesired to transplant into the host donor-derived material bearing onlyclass I histocompatibility antigens, it may be necessary to eliminateonly class I-reactive host T lymphocytes in Step 3. This could beaccomplished by infusing, in Step 2, donor antigens bearing only class Iantigenic determinants. On the other hand, additional donor antigenicdeterminants may be present in the infusion of Step 2 even though hosttolerance to these additional antigenic determinants may not benecessary. Thus, elimination of class I- and class II-reactive host Tlymphocytes by infusion of donor antigens bearing class I and class IIantigenic determinants may be performed even if the later transplanteddonor material bears only Class I antigenic determinants.

The donor antigens infused in Step 2 can be viable hematopoietic stemcells from a non-syngeneic donor. The donor hematopoietic stem cellsgenerally are not T cell depleted, although use of T cell depleted donorhematopoietic stem cells in Step 2 is also within the scope of thisinvention. Donor hematopoietic stem cells for use in Steps 2 and/or 4may be obtained, for example, by direct extraction from the bone marrowor from the peripheral circulation following mobilization from the bonemarrow. The latter can be accomplished by treatment of the donor withgranulocyte colony stimulating factor (G-CSF) or other appropriatefactors that induce mobilization of stem cells from the bone marrow intothe peripheral circulation. The mobilized stem cells can be collectedfrom peripheral blood by any appropriate cell pheresis technique, forexample through use of a commercially available blood collection deviceas exemplified by the CS3000 Plus blood cell collection device marketedby the Fenwal Division of Baxter Healthcare Corporation. Methods forperforming apheresis with the CS 3000 Plus machine are described inWilliams et al., Bone Marrow Transplantation 5: 129-133 (1990) andHillyer et al., Transfusion 33: 316-321 (1993). Alternative sources ofstem cells include neonatal stem cells (e.g., cord blood stem cells) andfetal stem cells (e.g., fetal liver of yolk sac cells). Stem cells thathave been expanded in vitro with a mixture of hematopoietic cytokinesalso may be used. Other useful stem cell preparations include stem cellsthat have been transduced with genes encoding donor-type MHC class I orclass II molecules, as well as stem cell preparations containing stemcells and/or T cells transduced with herpes simplex thymidine kinase orother “suicide” genes to render the mature T cells sensitive toganciclovir or other appropriate drugs in the event of severe GVHD.

(iii) Step 3

With respect to Step 3, “elimination” of the proliferatingdonor-specific host T lymphocytes as used herein includes host Tlymphocyte inactivation or tolerization as well as host T lymphocytedeath. Examples of lymphocytotoxic agents useful in Step 3 include Cy,melphalan and methotrexate. Cy, for example, is a short acting cytotoxicdrug known for its ability to kill lymphocytes, especially cells thatproliferate in response to antigenic stimulation (Bach J F, Amsterdam:North-Holland (1975); Aisenberg et al., Nature, 213:498 (1967); Paul WE, Fundamental Immunology. New York: Raven, (1984)). Cy can alsofacilitate activation of antigen-specific T cell suppressors responsiblefor maintenance of the tolerant state. Chernyakhovskaya et al.,Transplantation, 38:267 (1984); Maeda et al., Transplantation, 57:461(1994). Other agents known to eliminate proliferating T cells inresponse to donor antigenic stimulation may also be used, includingmonoclonal antibodies against activation markers of T lymphocytes suchas anti-CD25, anti-DC69 and anti-Ia/DR antibodies. Alloreactive host Tcells may be tolerized, rather than killed, by using agents that blockco-stimulation in conjunction with activation, since T cell engagementwith antigen without a second signal provided by co-stimulation resultsin tolerance. Such tolerizing agents include without limitationCTLA4-Ig, anti-B7.1 or anti-B7.2, anti-CD28, and antibodies againstadhesion molecules such as anti-LFA1, anti-CD44 and similar agents. Iftolerizing agents are used, steps 2 and 3 can be performedsimultaneously.

(iv) Step 4

In order to ensure an acceptable state of stable, mixed chimerism withrelatively high numbers of circulating donor cells, donor hematopoieticstem cells are administered to the host following performance of Step 3.This infusion of donor stem cells (Step 4) is derived from the samedonor, or from a donor genetically identical to that providing theantigens for Step 2. Hematopoietic stem cells from bone marrow, frommobilized peripheral blood populations, or other stem cell preparationsas described above (e.g., cord blood stem cells), may be used. Thenumber of stem cells administered in Step 4 can vary depending on the Tcell content of the stem cell preparation. If the preparation is not Tcell-depleted, then relatively small numbers of stem cells generally areadministered. If the stem cell preparation is T cell-depleted, thenlarger numbers of stem cells can be administered since there is no riskof GVHD.

The donor hematopoietic stem cells of the second infusion may or may notbe T cell depleted, depending on the immunologic disparity between thedonor and recipient, the intensity of immunosuppression given in Step 1and the degree of chimerism desirable in view of the immunogenicity ofthe graft. When higher fractions of sTLI (4-12), or otherimmunosuppressive agents providing equivalent immunosuppression, areused in the immunosuppressive regimen of Step 1, the second infusioncomprising donor hematopoietic stem cells typically is T cell depletedto control for GVHD. When Step 1 involves little immunosuppression (forexample, 1-3 fractions of sTLI), or when Step 1 is eliminatedaltogether, the infusion of donor hematopoietic stem cells in Step 4typically is not T cell depleted. If not T cell depleted, the donor stemcells provided in Step 4 can be infused in graded increments over aperiod of weeks or several months, while monitoring for signs of GVHD.

In mouse experiments reported below, the mice received sTLI of 0-6fractions of 200 cGy/fraction (Step 1). The donor-reactive T cells ofthe host were activated (Step 2) by injecting non-T cell depleted donorBMC (3×10⁷ cells). The activated host T cells were subsequentlyeliminated (Step 3) by a non-myeloablative dose (200 mg/kg or 3 doses of60 mg/kg) of Cy. Mixed chimeras with low levels (e.g., 7%-20%) of donorcells in the blood were predominant after the Cy treatment. Induction ofhigher levels of hematopoietic mixed chimerism (e.g., >20% of donorcells in blood) was achieved by administering (Step 4) a second infusioncomprising donor hematopoietic stem cells, allowing life-long survivalof donor skin allografts.

In mice treated with 6 doses of TLI, Step 4 was required to achieve alevel of tolerance permitting acceptance of full thickness skinallografts. It is well known that full-thickness skin presents the moststringent test for donor-specific tolerance. Skin allograft acceptancecan be accomplished only in stable chimeras (Maeda et al., J. Immunol.,150:753 (1993)) and success of skin acceptance may depend on the levelof donor-derived cells in the host's blood.

In further experiments reported below, deletion of all host-derived,donor-reactive T lymphocytes following Step 3 permitted rapidengraftment of even low numbers of donor stem cells (2-3×10⁶/mouse)administered in Step 4, which normally would not be sufficient forinduction of stable mixed chimerism. In parallel with this, the fullanti-donor unresponsiveness induced following Step 3 also resulted inexquisite sensitivity to donor T lymphocytes, leading to lethal GVHD.Hence, whenever donor-reactive host T cells are effectively depleted,elimination of immunocompetent T lymphocytes from the hematopoietic stemcell preparation administered in Step 4, or use of lifespan-limitedlymphocytes (e.g., carrying suicide genes), is crucial for prevention ofGVHD. Due to the selective deletion of donor-reactive host lymphocytes,even 2-3×10⁶ BMC (T cell-depleted), administered following Step 3,engrafted and converted host mice into stable mixed chimeras withrelatively high levels (20%-50%) of donor-derived hematopoietic cells inthe blood.

T cell depletion of donor stem cell preparations has been known toincrease the risk of graft rejection. Thus, inoculation with extremelylarge numbers of donor stem cells has been mandatory for engraftment ofT cell depleted BMC, especially in recipients conditioned with previousnon-myeloablative protocols. Truitt et al., Blood 77, 2515-2523 (1991);Reisner et al., Immunol. Today 16, 437-440 (1995); Bachar-Lustig et al.,Nat. Med. 12, 1268-1273 (1995). The ability to induce engraftment usinglow numbers of donor hematopoietic stem cells (T lymphocyte depleted) isa significant advantage of the present protocols. This is due to theimproved acceptance of donor hematopoietic stem cells on the one handcombined with a reduced risk of GVHD that would otherwise follow fromthe use of higher stem cell inocula, on the other. Notably, animalsnon-specifically immunosuppressed by sTLI and Cy (without infusion ofdonor-antigens prior to Cy) were shown to reject T lymphocyte depletedBMC. Thus, the present data clearly show the advantage of donor-specifictolerogenic conditioning in comparison with non-specificimmunosuppression approaches, while avoiding potentially hazardous highdoses of TBI. GVHD can also be controlled using the tolerogenic agentsdescribed above for use in step 3. In addition, antibodies to CD52, CD40ligand, CD40, IL-2 receptors (e.g., CD25) can be used to modulate GVHD.It is understood that the term antibody applies both to nativeantibodies, or as functional fragments of antibodies (e.g., Fab,F(ab′)₂, or Fv fragments). Furthermore, they can be used asimmunotoxins, e.g., conjugated with toxins such as Pseudomonas toxin ordiphtheria toxin, ricin, or a radionuclide, e.g., ¹²⁵I or ¹³¹I.

Interestingly, life-long tolerance to full thickness donor-derived skingrafts was also accomplished in recipients who were not subjected toStep 1 and in recipients in which Step 1 involved only a single fractionof TLI (200 cGy). Thus, a balance exists between the intensity of theconditioning of the host and the susceptibility of the host to GVHDinduced by the presence of donor-derived T cells: recipients of a singlefraction of sTLI could resist GVHD induction by a large inoculum ofnon-T cell depleted donor BMC whereas the sensitivity of the hosts toGVHD was increased in recipients conditioned with 6 fractions of sTLI.Hence, a second infusion (Step 4) comprising non-T cell depleted donorBMC could be used relatively safely in recipients of 1 dose of sTLIwhereas T cell depletion of the second infusion was mandatory inrecipients of 6 fractions of sTLI. The sensitivity of the 6×sTLIrecipients to develop GVHD was likely due to inability to veto thehost-reactive donor cells due to effective depletion of all host Tcells.

Tolerant mixed hematopoietic chimeras generated by the tolerogenictreatment described herein remain immunocompetent to third party grafts.In experiments described below, all tolerant B6→BALB/c chimeras thataccepted B6 skin allografts rejected non-relevant CBA skin grafts within16-20 days (n=11). Thus, tolerance induction neither eliminated norimpaired normal reactivity by the host immune system retained in themixed chimera. This is an important advantage of the method, sincerecipients are not immunocompromised due to transient loss of allhost-derived immune cells, which is otherwise unavoidable when chimerasare comprised of 100% donor cells following TBI. A patient who retains ahost-derived immune apparatus with memory cells is in a better positionto resist primary and secondary infections. This retained resistance tointercurrent infections, particularly to viral agents infecting hosttarget cells, is of crucial importance. This is because the donorhematopoietic cells may be MHC disparate and, therefore, incapable ofproviding immune protection against virally-infected host tissues.

The above-described tolerogenic treatment may be employed to inducetransplantation tolerance across xenogeneic barriers. Xenogeneic skintransplantation may be considered the most stringent test fordonor-specific tolerance. As described below, the present inventor hassucceeded in inducing permanent tolerance in mouse-to-rat skin grafts.The same donor-specific tolerance induction protocol presented herewithcan be applied to xenogeneic transplantation in humans. The xenogeneicgraft (e.g., pancreatic islets) may be taken from non-human mammals andtransplanted into humans.

A tolerogenic treatment for xenogeneic transplantation may be performedas follows. sTLI (Step 1) is carried out, followed by an infusion (Step2) of xenogeneic donor antigens, for example, BMC. Subsequently, atleast one non-myeloablative dose of lymphocytotoxic or tolerizing agentis administered (Step 3). If necessary, the lymphocytotoxic agent can beadministered in multiple low doses over several days. Administration ofthe lymphocytotoxic agent is followed by a infusion of a preparationcomprising T cell-depleted donor hematopoietic stem cells (Step 4). Thestem cells may be obtained from the blood or bone marrow of an adultdonor. Alternatively, partially immunocompetent cord blood cells may beused, or even fetal stem cells obtained from the liver or yolk sac ofembryos. Stem cells that have been expanded in vitro with a mixture ofhematopoietic cytokines also may be used. Administration of stem cellsin Step 4 leads to engraftment of the xenogeneic donor stem cells andpermanent transplantation tolerance of the host to donor derived organs.In an alternative embodiment, xenogeneic transplantation may beperformed without administration of a non-myeloablative regimen (Step 1eliminated) and with the second infusion (Step 4) comprising non T celldepleted donor hematopoietic stem cells.

(v) Short Protocol for Induction of Transplantation Tolerance

In regard to use of the tolerogenic method of the invention innon-syngeneic organ, tissue or cell transplantation, the results of theexperiments described in Example 14 are of great importance. In theseexperiments, it was found that allogeneic grafts (bone marrow stroma orhearts) could be implanted without significant rejection on the same day(day 0) as a single dose of TLI (as step 1) and donor bone marrow (asstep 2), followed on day 1 by Cy (as step 3) and on day 2 by a seconddose of bone marrow (as step 4). This method was designated the “shortprotocol” in order to differentiate it from the longer protocols used insome other experiments. Thus, in such longer protocols, heart or skingrafts were, for example, implanted 20 days after cyclophosphamidetreatment (step 3) (Example 5) and 19 days after the second bone marrowinjection (step 4) (Examples 2, 3, 4, 6, 7, and 9). Furthermore, inanother long protocol, sTLI, as step 1, was given over a period of 6days (e.g., Example 3). It is hypothesized that, in the short protocol,the grafted organ acts together, and possibly in synergy, with the bonemarrow given on day 0, as antigen. It is possible, in addition, that thebone marrow given on day 0 may not be necessary for establishment oftolerance. This method of the invention is however not limited by aparticular mechanism of action.

The success of the short protocol broadens the applicability of thetolerogenic approach of the invention for human transplantation. Forexample, in the case of cadaveric transplantation, donor bone marrow (orany other tissue that can be used for step 2) is not normally availablesignificantly in advance of the availability of the relevant organ(e.g., kidney, heart, liver, or lung), tissue (e.g., skin, bone, muscle,or cartilage), or cells (e.g., hepatocytes or pancreatic islet cells).Using the short protocol, bone marrow can be harvested from the donor atthe same as the organ and both can be given to the recipient on the sameday as non-myeloablative conditioning (e.g., TLI) (day 0).Simultaneously, bone marrow cells can be frozen and stored for use instep 4, either on day 2 and/or on subsequent days. In addition tobroadening the applicability of the tolerogenic methodology of theinvention for cadaveric allo-transplantation, the short protocol canalso, for the same reasons, greatly simplify the logistics ofliving-related donor or xenogeneic donor transplantation.

(vi) Articles of Manufacture

Also included in the invention are articles of manufacture includingpackaging material (e.g., a cardboard box) containing a biological cellcontainer e.g., a blood bag such as a semipermeable blood bag that canbe used for culturing cells). The biological container can contain acomposition that includes hemopoietic stem cells and the packagingmaterial can include a package insert or a label indicating that thecomposition can be used as the antigen in step 1 and/or as the source ofhematopoietic stem cells for step 4 of the tolerogenic protocol.

(vii) Chimeras

In another embodiment, the invention involves a method of making anon-human mammal/human hematopoietic chimera. The method comprisesmaking a non-human mammal tolerant to antigens originating from a humandonor, using the non-myeloablative tolerogenic treatment describedherein. That is, the non-human mammal functions as the “host mammal” inthe protocols described above, and a human being is the “donor.” Forexample, a rodent can be tolerized to human cells, tissues and organs byemploying Steps 1-4 of the disclosed tolerogenic protocol to produce amixed chimera rodent permanently engrafted with human hematopoieticcells. It is known that such hematopoietic engraftment is possible evenbetween disparate species. For example, it has been demonstrated thathuman hematopoietic cells can engraft in mice. See, for example, Marcuset al., Blood 86: 398-406 (1995). In those cases where survival andfunctioning of human hematopoietic cells is less than optimal innon-human mammalian hosts, it is possible to provide the host mammalwith human hematopoietic cytokines in order to ensure engraftment of thehuman cells.

There are numerous uses for such chimeric animals. For example, sincethe host mammals have been tolerized to the human donor, it is possiblefor human tissues, e.g., tumors or HIV-infected hematopoietic cells, tobe transplanted into and accepted by these rodents in order to producerodent models of human disease. Thus, these non-human mammal/humanchimeras may be used to study biological phenomena related to humandisease, including testing of new drugs.

Production of non-human mammal/human hematopoietic mixed chimeras is ofeven greater significance for those non-human mammalian species targetedas potential sources of cells, tissues and organs for transplantationinto human patients. For example, it is widely recognized that pigs area potential useful source of tissues and organs for transplantation intohumans. Such porcine materials are subject to an immediate, “hyperacute”rejection response when transplanted into human patients, as well as tolonger-term immune-mediated rejection by the human host. Pigs are beinggenetically engineered or otherwise treated to protect tissues andorgans of such pigs from being hyperacutely rejected when transplantedinto a human patient. This can be accomplished, for example, byproviding the pigs human genes encoding human complement regulatoryproteins, or by “knocking out” the genes responsible for production ofpig antigens recognized by preformed xenoantibodies present in allhumans. See, for example, PCT/US96/15255 and PCT/IB95/00088.

A “two-way” variation of the present tolerogenic protocols can beapplied to such genetically engineered pigs as well as to other donormammals to allow for ready transplantation of xenogeneic donor cells,tissues and organs into humans. For example, in a preliminarytolerization procedure, a human patient can function as an initial“donor” to provide antigens and hematopoietic stem cells to a “host” pigin the 4-Step protocol described above. As a result, the pig istransformed into a pig/human hematopoietic mixed chimera, with the pig'shematopoietic cells being tolerized to the human patient's cells,tissues and organs. Following this, the roles of the human patient andpig are reversed, with the pig becoming the donor and the human patientbecoming the host in the 4-Step protocol. That is, the pig'shematopoietic cells, with T cells tolerant of the human patient, may beused in the 4-Step protocol for transformation of the human patient intoa human/pig hematopoietic mixed chimera. The human patient is then ableto accept cells, tissues and organs from the pig, for the reasonsdiscussed above. The crucial advantage is that all of this can beaccomplished while avoiding the risk of xenogeneic GVHD engendered byimmunocompetent T cells of the pig, since the pig's T cells were madetolerant to the patient in the preliminary tolerization procedure. Thus,assuming the hyperacute rejection response can be overcome in other ways(e.g., genetic engineering of the animal providing the transplantedmaterial), the present invention allows for xenogeneic transplantationof cells, tissues and organs into humans without the need for long-termimmunosuppression. As an alternative to tolerizing the pig to the humanpatient's antigens, the pig's cells can be isolated and the tolerizationcan be performed in vitro, using standard tissue culture techniquesfamiliar to those in the art. The human antigens can be cellular ornon-cellular, as in step 2. They can be, for example, any of thehematopoietic cells described herein. Furthermore, the procedure,whether in vivo or in vitro, can be carried out using allogeneichost/donor mammalian (e.g., human, non-human primate, pig, rat, mouse,rabbit, or guinea pig) combinations as well as xenogeneic combinations.

In addition, tolerization of donor cells need not be antigen-specific.It can involve depletion of, for example, substantially all T cells inthe bone marrow to be used in step 4, with the retention of stem cellsand, optionally, other functional cell subsets (e.g., NK cells) that canhave therapeutic benefit for a host subject suffering from a relevantdisease (e.g., cancer). Methods for non-specifically depleting T cellsfrom a hematopoietic cell sample include those described above for steps1 and steps 3 of the tolerization protocol or those described below forsteps A and C of the protocol used to deplete cells to be used for celltherapy. Experiments using such a non-specific in vitro depletingprotocol are described in Example 15.

(viii) Cell Compositions

Another aspect of the invention is a hematopoietic cell compositionderived from a host treated with the donor-specific tolerogenictreatment described above. The cell composition compriseshost-originating lymphocytes and donor-originating lymphocytes. Theproportion of donor-originating lymphocytes may vary. Preferably, thedonor-originating lymphocytes comprise about 5% to about 50% of thelymphocyte composition. Most preferably, the donor-originatinglymphocytes comprise about 20% to about 50% of the lymphocytecomposition. The hematopoietic cell composition is specifically depletedof donor-specific, host-originating lymphocytes. “Depleted” in thiscontext refers to reduction in numbers of T lymphocytes or reduction inT lymphocyte function sufficient to eliminate or significantly reduceanti-donor responses in the host and thus to reduce the risk of GVHD ifthe composition is administered to the donor. In a related aspect, theinvention involves a method of making the above described hematopoieticcell composition. The method includes subjecting a host mammal to thetolerogenic treatment described above using an allogeneic or xenogeneicdonor. After tolerogenic treatment, the method involves isolating acomposition of hematopoietic cells from the host. This cell compositioncontains host and donor-originating hematopoietic cells, but is depletedof donor-specific host-derived T lymphocytes.

B. Cell Therapy Protocols

Completion of the tolerogenic treatment protocols can provide a platformfor subsequent allogeneic cell therapy with donor lymphocyte infusionsin cancer patients and in other patients with malignant andnon-malignant diseases requiring bone marrow transplantation, sincedonor cells accepted by a tolerant host may induce graft-versus-leukemia(GVL) or graft-versus-tumor (GVT) effects. Such non-malignant diseasesinclude without limitation a plastic anemia, genetic diseases resultingin enzyme deficiencies, and diseases caused by deficiencies inwell-defined products of hematopoietic stem cells, such as osteoclastdeficiency in infantile osteopetrosis and deficiencies in B cells and Tcells in congenital and acquired immune-deficiency syndromes. Donors ofcells for use in allogeneic cell therapy can be MHC (e.g., HLA inhumans) incompatible or MHC identical with the host. Where MHCincompatible, the donor and host can share no MHC class I or class IIalleles. Alternatively, they can share 1 or more (e.g., 2, 3, 4, or 5)MHC class I and/or class II alleles. Where donor and host are MHCidentical, they can be incompatible at MiHL. They will thus preferablynot be monozygotic twins. Allogeneic cell therapy is described, forexample, in PCT publication no's. WO 95/24910 and WO 96/37208.

In allogeneic cell therapy, an anti-tumor or other anti-hosthematopoietic cell effect is achieved by administering allogeneicperipheral blood lymphocytes to the host, either alone or in combinationwith a T cell activator. Alternatively, allogeneic peripheral bloodlymphocytes are “pre-activated” in vitro by a T cell activator such asinterleukin-2 (IL-2) and then administered either alone or incombination with the same or different T cell activator. Preferably, oneor more infusions of about 10⁵ to about 10⁹ cells/kg of allogeneicperipheral blood lymphocytes, including well-defined lymphocyte subsets,are administered. When preceded by the tolerogenic treatment describedherein, these infusions of allogeneic lymphocytes are carried out with amuch reduced chance of rejection of the anti-cancer effector cells,which need to become engrafted in the host. In addition, the risk ofGVHD is reduced or eliminated by residual hematopoietic cells of thehost and, if necessary, relatively late infusion of donor lymphocytes.

Allogeneic cell therapy following the tolerogenic treatment protocolsdescribed herein can be valuable not only in the context of cancer andother diseases, but also when it is desired to adoptively transferimmunity to infectious agents from the donor to the host. Thus, if adonor used in the tolerogenic protocols described herein is immune to aninfectious agent (e.g., hepatitis B; see Ilan et al., Hepatology 18:246-52 (1993)), this immunity can be transferred to a host by infusinglymphocytes from the donor to the host following completion of thetolerogenic protocols. Alternatively, the stem cell preparation infusedin Step 4 of the tolerogenic protocols can itself provide the adoptivetransfer of immunity, since stem cell preparations may containimmunocompetent lymphocytes.

From the above descriptions of depletion-mediated allogeneic andxenogeneic tolerance induction, particularly the discussion of methodsfor depleting hemopoietic cells (e.g., pig hemopoietic cells) ofhost-specific activity prior to their use is step 4 of the tolerizingprotocol, it is clear that cell therapy can be performed using cellsfrom individuals that are xenogeneic as well allogeneic to the host(i.e., recipient of cell therapy). In xenogeneic cell therapy, the samecompositions of lymphocytes, with or without one or more T cellactivators, e.g., IL-2, interferon-γ (IFN-γ), granulocyte-macrophagecolony stimulating factor (GM-CSF), or interleukin-12 (IL-12), describedabove for allogeneic cell therapy, can be used. Moreover, the cells canbe pre-activated, either in vitro or in vivo, prior to administration tothe host. The cells to be used for cell therapy can be obtained from theblood, spleen, lymph nodes, or any other source of lymphoid cells, orthey can obtained from a hematopoietic cell source, e.g. bone marrow orfetal liver.

(i) Depletion of Cells to be used for Cell Therapy

Cell therapy can be given by administering unselected lymphoid cells. Ingeneral, however, prior to administration, the lymphoid cells will bedepleted of T cells reactive to the recipients antigens or ofresponsiveness of T cells to the host's antigens. In the followingdescription, the word “deplete” (or “depletion”) refers to decreasingthe numbers of T cells with responsiveness to antigens of the secondindividual and/or decreasing the responsiveness of such T cells.Depletion can be by decreasing the number of responsive lymphocytes orthe responsiveness of the lymphocytes by 20%, 30%, 40%, 50%, 80%, 90%,95%, 98%, 99% or even 100%.

In the above description of depleting donor hemopoietic cells ofhost-specific activity, the host/donor terminology was reversed. In thefollowing discussion of methods to deplete lymphoid cells to be used forcell therapy, in order to avoid confusion as to the “host” and the“donor”, the individual that is the source of the cells to be used forcell therapy will be referred to as the “first individual” and theindividual that is to receive the cell therapy (e.g., a human cancerpatient) will be referred to as the “second individual”. Furthermore,the phrase “non-syngeneic” will be understood to cover both allogeneicand xenogeneic.

Methods of depleting lymphoid cells prior to their use for cell therapybasically involve the use of one or more of steps 1-4 described abovefor tolerizing a host against antigens of a donor. In order, however, todifferentiate the steps used for depleting lymphoid cells to be used forcell therapy from those used for tolerizing a host mammal prior totransplantation, the steps used for depletion of lymphoid cells to beused for cell therapy are designated “steps A-D”:

Step A: Expose lymphoid cells of a first individual to anon-myeloablative regimen sufficient to decrease, but not eliminate, thefirst individual's functional T lymphocytes.

Step B: Contact the lymphoid cells of the first individual with antigensof a non-syngeneic second individual.

Step C: Substantially eliminate T lymphocytes of the first individualthat are responsive to the antigens of the second individual bydelivering a non-myeloablative dose of a lymphocytotoxic or tolerizingagent to the lymphoid cells.

Step D: Administer a preparation of hemopoietic stem cells obtained fromthe second individual to the first individual.

Essentially the same methods for performing steps 1-4 described abovecan be used to carry out steps A-D, except that some or all of the stepscan be carried out in vitro using tissue culture methods familiar thosein the art. Naturally, step D is always in vivo and can only be includedwhere the entire depleting procedure is performed in vivo.

The lymphoid cells can be depleted using either (i) step A alone, (ii)step B alone, (iii) steps A and B, (iv) steps B and C, (v) steps B, C,and D, (vi) steps A, B, and C, or (vii) steps A, B, C, and D.

A method of depleting lymphoid cells of a first individual of reactivityagainst a second individual can involve, for example, harvestinglymphoid cells from the first individual and carrying out steps A-C invitro. Alternatively, after carrying out step A in the first individual,the lymphoid cells can be isolated from the first individual and steps Band C carried out in vitro. In another embodiment, steps A and B can becarried out in the first individual, the lymphoid cells can be isolatedfrom the first individual, and step C carried out in vitro. In addition,all the steps can be carried out in vivo, optionally including step D.

(ii) Step A

Step A decreases the total number of T cells in the cells to be used fortherapy and is optionally used. Steps B and C are used to deplete onlythose cells or that T cell reactivity specific to donor antigens. Asindicated by the experiments in Examples 10-13 below, step C, in somecases (e.g., where cancer cells are used as antigens), can be excludedsince step B, can be sufficiently tolerogenic.

(iii) Step B

The same sources of antigen used in step 2 can be used in step B andthey can be obtained by the same methods. In addition, however, cancercells can be used. As indicated by the experiments in Examples 10-13below, lymphoid cells depleted using cancer cells for step B can beparticularly useful for non-syngeneic cell therapy of a mammal withcancer, e.g., a human cancer patient. The experiments, which werecarried out using mouse strains (X and Y) which differed either at bothMHC and Minor Histocompatibility Loci (MiHL) or at MiHL only, indicatethat lymphoid cells from a mouse of strain X exposed to cancer cells ofstrain Y, while being substantially depleted of graft-versus-hostactivity against antigens of strain Y, have enhanced anti-cancertherapeutic efficacy when transferred together with cancer cells to miceof strain Y. The therapeutic activity of the strain X lymphoid cellsexposed to strain Y cancer cells was significantly greater than that ofstrain X lymphoid cells exposed to strain Y lymphoid cells, which, atleast in the case of a MiHL incompatibility alone, was significantlygreater than that of normal, unexposed, strain X lymphoid cells.Furthermore, in a mouse combination in which strains X and Y differ atboth the MHC and MiHL, while lymphoid cells from normal, unexposedstrain X mice displayed lethal GVH activity when transferred to hostanimals expressing strain Y antigens, lymphoid cells from strain Xanimals exposed either to cancer cells of strain Y or to lymphoid cellsof strain Y were substantially depleted of the GVH activity. On theother hand, in a mouse combination in which strains X and Y differ atthe MiHL only, exposure of strain X animals to cancer cells of strain Ydepleted the strain X lymphoid cells of strain Y-specific GVH activityrelative to lymphoid cells from normal, unexposed strain X animals butexposure of strain X animals to lymphoid cells from strain Y animals didnot have an effect on the GVH activity of strain X lymphoid cells.

(iv) Use of Tumor Cells for Step B

These experiments point to a novel form of non-syngeneic cell therapy ofmammalian, and in particular human, cancer, in which the protocol usedto deplete the lymphoid cells to be used for therapy of GVHD activityalso results in enhanced anti-cancer therapeutic efficacy in thelymphoid cells. A subject to be given cell therapy with lymphoid cellsof another histoincompatible subject could be treated by any or all ofsteps 1-4 above in order to establish non-reactivity (tolerance) to theantigens of the other subject. Alternatively, the cell therapy could begiven without any of these steps. It could be given, for example, to apatient that for unrelated reasons (e.g., HIV AIDS, geneticimmunodeficiency, chemotherapy, or radiotherapy) is significantlyimmunologically depleted (i.e., with a functional T cell populationdepleted by greater than 80%, 90%, 95%, or 99%). The lymphoid cells usedfor the therapy could be from an unrelated MHC compatible orincompatible human, a related MHC compatible or MHC incompatible human,or an individual of another species, e.g., a pig or a non-human primate.The lymphoid cells will preferably not be from a syngeneic individual(e.g., a monozygotic twin).

The cancer cells that can be use for step B are preferably of the typepresent in the subject to be given the cell therapy, e.g., leukemia,lymphoma, breast cancer, lung cancer, gastrointestinal cancer, melanoma,or genitourinary cancer, and thus will express the same antigens orcross-reactive antigens. However, it remains possible that the enhancedanti-cancer therapeutic efficacy of non-syngeneic lymphoid cellsdepleted by exposure to tumor cells is not antigen specific and thatcells depleted by exposure to a wide variety of tumors, will be haveanti-tumor effects against the tumor harbored by a given subject. Thusstep B can also be performed using, as a depleting agent, tumor cells ofa type unrelated to that of the subject.

Cancer cells to be used for Step B will preferably express MHC molecules(at least one MHC class I or MHC class I molecule) of the patient. Morepreferably, they will be derived from the patient. Tumor cells from allsubjects, including human patients, will be obtained by standard methodsknown to those in the art. In the case of non-solid, hematologicalcancers, the cells can be isolated or enriched from the blood, bonemarrow, spleen, lymph nodes or other lymphoid tissue of the subject. Inthe case of solid malignancies, tumor cell suspensions can be obtainedby disruption of solid tumor tissue removed by surgical excision ofeither the primary tumor or metastases. Where tumor cells of the patientare not available or are not available in sufficient quantity, it ispossible that established tumor cell lines can be used. Such tumor celllines can endogenously express the MHC molecules of the patient or theycan have been stably transfected with and express genes encoding suchmolecules. If necessary, a plurality of such transfected lines, eachexpressing, either endogenously or due to stable transfection, the MHCmolecules of the patient. Subcellular fractions (e.g., cell lysates ormembrane fractions) of any of the above tumor cell preparations can alsobe used as antigen for step B. Furthermore, subcellular fractions, orisolated tumor antigens can be presented to donor T cells in associationwith antigen presenting cells (e.g., dendritic cells, macrophages,monocytes, or B lymphocytes), which can be freshly prepared orprecultured. The antigen presenting cells can express MHC molecules ofthe patient, and preferably will be derived from the patient. Suchantigen presenting cells can, for example, be pulsed with tumor cellextracts or peptides derived from tumor-associated polypeptide antigensprior to their use in step B. Alternatively, the antigen presentingcells can be transduced or stably transfected with expression vectorsencoding full-length tumor-associated polypeptide antigens or peptidescorresponding to subregions of such antigens. In addition, cell hybridsformed by fusion of tumor cells and antigen presenting cells can be usedas the activating antigen for step B of the method.

It is envisaged that, where the second individual (i.e., the recipientof cell therapy) is a human patient and step B is to be carried out invivo, the first individual (i.e., the source of the non-syngeneic cells)will likely either be a relative of the patient or an individual ofanother species. However, whether in vivo, in vitro, in a relative, orin an individual of another species, proliferation of the tumor cellsused as antigen can be substantially eliminated by prior exposure of thetumor cells to a sufficient dose of an anti-proliferative agent, e.g.,ionizing radiation or mitomycin-C. Alternatively, the above-mentionedsubcellular fractions can be used.

(v) Non-antigen-specific Methods of Depleting Cells

Protocols for non-specifically depleting hematopoeitic cells ofresponsive T cells can involve the use of any of the methods describedabove for step A without any of steps B-D. In addition, any of a widevariety of methods known in the art (e.g., use of magnetic beads, lyticcomplement, or flurorescence activated cell sorting) employing single orcombinations of antibodies (monoclonal or polyclonal) that bind to Tcells (or cells other than T cells) can be used to remove T cells. Suchprotocols can employ single or multiple (e.g., 2,3,4,5,6,8,10 or 12)treatments and one or more of the methods can be used. Furthermore, theycan be performed either in vivo or in vitro. Such protocols will resultin elimination of substantially all (e.g., greater than 80%, preferablygreater than 90%, more preferably greater than 95%, and most preferablygreater than 99%) of the T cells in the hematopoietic cell population.It is noted, as described above, that such protocols can also be used todeplete donor cells of reactivity to the host prior to use in step 4 ofthe tolerization method of the invention.

An example of such protocol is provided in Example 15.

(vi) Cell Compositions

The instant invention also encompasses cell compositions containinglymphocytes obtained from a first individual that have been depleted ofreactivity to the antigens of a second individual. The depletion willpreferably have been performed by one of the methods described aboveinvolving the indicated possible combinations of steps A-D. Such methodsinclude the use of both cellular and non-cellular antigens for step Band the optional use of step D (i.e., administering bone marrow cellsderived from the second individual to the first individual). Thus thecompositions can contain 20%-100%, 30%-100%, 50%-100%, 70%-100%, or90-100% of cells derived from (i.e., endogenous to) the firstindividual. The cells of the compositions will be suspended in aphysiological solution, e.g., a saline solution, and provided in anappropriate container, e.g. a blood bag, a semipermeable blood bag, atissue culture receptacle such as a tissue culture flask, or abioreactor. The lymphocyte source (e.g., blood, bone marrow, spleen, orlymph nodes) can be obtained from the first individual, at theappropriate stage of depletion, and purified or enriched by methodsfamiliar to those in the art.

It is envisaged that “batches” of such cell compositions can be made andstored by a supplier using lymphocytes from appropriate human ornon-human (e.g., pigs and non-human primates) donors. The lymphocytescan be depleted, using as step B, for example, single or combinations oftumor cell lines stably transfected with and expressing combinations ofall known MHC genes. In this way, batches of lymphocytes would bederived, each being tolerant to a different set of HLA antigens. Thesecustom pretolerized batches of lymphocytes can then be supplied to apractitioner (e.g., an oncologist) whose patient is in need of thetherapy after communication of the patient's HLA haplotype to thesupplier.

If it is proposed to carry out the tolerogenic method of the inventionprior to non-syngeneic cell therapy, the practitioner can be suppliedwith a source of antigenic material (e.g., hemopoietic cells) for use instep 2 of the tolerogenic protocol. This antigenic material can bederived from the individual from which the lymphocytes used for celltherapy were obtained, or from an individual syngeneic with theindividual. Alternatively, either cells (e.g., non-malignant hemopoieticcells) stably transfected with appropriate HLA genes or cell extracts ofeither malignant or non-malignant cells similarly transfected can beused.

(vii) Methods of Cell Therapy

Also within the scope of the invention are methods of treatment thatinclude administration of the above cell compositions to a subject,preferably a human patient. As indicated above, such subjects mayoptionally have been subjected to one of the described tolerogenicregimes. Patients to which the treatment can be given include cancerpatients (e.g., those with the above-listed tumors), patients sufferingfrom infectious diseases such as HIV AIDS or hepatitis B or C, geneticdiseases associated with protein (e.g., hemoglobin or an enzyme)deficiencies or abnormalities, aplastic anemia, congenitalimmunodeficiencies, and autoimmune diseases such as rheumatoidarthritis, multiple sclerosis, insulin-dependent diabetes mellitus,lupus erythematosus, and myasthenia gravis.

(viii) Articles of Manufacture

Also encompassed by the invention are articles of manufacture includingpackaging material (e.g., a cardboard box) containing a biological cellcontainer such as those listed above. The biological container containsany of the cell compositions described above and the packaging materialcan include a package insert or a label indicating that the compositionis to be used in a method of treatment comprising administering of thecomposition to a mammal in need of the composition (e.g., patients withany of the diseases listed above).

The invention will be further understood with reference to the followingillustrative embodiments, which are purely exemplary, and should not betaken as limiting the true scope of the present invention as describedin the claims.

EXAMPLE 1 Materials and Methods

Animals

Inbred BALB/c (H-2^(d)), C57BL/6 (B6) (H-2^(b)), DBA/2 (H-2^(d), CBA(H-2^(k)), B10.D2 (H-2^(d)), SJL (H-2^(s)) and (BALB/c×C57BL/6)F₁ (F1)(H-₂ ^(d/b)) mice and Lewis rats were purchased from the HebrewUniversity Hadassah Medical School Animal Facility in Jerusalem, Israel,with breeding pairs originating from Harlan-Olack, Bicester, UK. Two tothree month-old mice were used for the study. Mice were kept understandard conditions with food and water provided ad lib. Most of theexperiments were carried out in B6→BALB/c strain combination.

Total Lymphoid Irradiation (TLI)

Mice were anesthetized and then positioned in an apparatus designed toexpose the major lymph nodes, thymus, and spleen to ionizingirradiation, while shielding most of the skull, ribs, lungs, hind limbsand tail with lead, as previously described. Slavin et al., J. Exp.Med., 146:34 (1977). Radiation was delivered by a Phillips X-ray unit(250 kv, 20 mA) at a rate of 70 cGy/min, using a Cu 0.2-mm filter. Thesource-to-skin distance was 40 cm.

Tolerogenic Treatment

The basic protocol for conditioning prior to transplantation includedsTLI (1-6 daily exposures of 200 cGy) to a total dose of up to 1,200cGy, followed by intravenous inoculation with 3×10⁷ BMC on the day afterthe last TLI dose. Some mice (see Example 9) were not administered anysTLI. One day after BMC infusion, experimental mice were injected with200 mg/kg Cy (Taro, Israel) intraperitoneally. Cy was freshly dissolvedin sterile phosphate-buffered saline prior to injection. Modificationsof the Cy protocol to induce tolerance to xenografts are described inExample 8. A second infusion of donor BMC, after Cy, was alsoadministered in some of the mice.

Preparation of Bone Marrow and Spleen Cells

Single cell suspensions of BMC and spleen cells were prepared in PBS orRPMI 1640 medium supplemented with 100 μg/ml streptomycin and 100 U/mlpenicillin (Biological Industries, Beit Haemek, Israel). BMC wereinfused into the lateral tail vein in a total volume of 0.5 ml.

Preparation of Blood Cells for Infusion

Pooled fresh blood was collected into heparin-containing tubes(preservative-free). Each recipient was infused with 0.5 ml into thelateral tail vein.

T Cell Depletion of BMC with Monoclonal Antibodies

Monoclonal rat anti-mouse Thy1 antibodies (YTS 148.3, IgM and YTS 154.7,IgG2b) were obtained from Dr. H. Waldmann (Oxford University, UK). BMC(10⁷/ml) were incubated with YTS 148.3 antibody at a final dilution of1:200 for 40 min, washed and incubated with Low-Tox rabbit complement(Cedarlane, Canada) at a final dilution of 1:10 for an additional 60 minat 37° C., washed and injected intravenously into recipients. YTS 154.7antibody was used for depletion of T cells from BMC in vivo; BMC(3×10⁶/ml) were incubated with 750 μg, 150 μg or 30 μg of the antibodyfor 60 min at 4° C. and the mixture was injected intravenously intorecipients.

Skin Grafting

Skin grafting was carried out 20 days after completion of thetolerogenic treatment. A full-thickness skin graft measuring 1 cm×1 cmwas adjusted to the graft bed by 4 Thomas surgery clips (ThomasScientific, USA ). The panniculus carnosus was kept intact in the graftbed. The graft was considered to be accepted when hair of donor colorgrew on the soft flexible underlying skin, and rejected when donorepithelium was lost.

Implantation of Bone Marrow Plugs

The femora of B6 mice were freed of muscle and irradiated with 400 cGyin vitro to eliminate most of the hematopoietic cells. Marrow plugs weremechanically pressed out of the femur canal with a mandrin and 2 plugswere implanted under the left kidney capsule of each recipient, asdescribed in Chertkov et al., Rad. Res. 79, 177-186 (1979).

Heterotopic Heart Grafting

Hearts of 1-2 day old B6 mice were transplanted into the ear skin pocket20 days after tolerogenic treatment, according to the methods ofChernyakhovskaya et al., Transplantation, 29:409 (1980). An ECG wasfirst recorded two weeks after grafting and thereafter at weeklyintervals.

Polymerase Chain Reaction (PCR)

PCR was carried out on material derived from blood samples as describedpreviously. Pugatsch et al., Leukemia Res. 17, 999-1002 (1993). Briefly,blood samples were lysed in distilled water and centrifuged at 12,000×g.Supernatants were discarded and 50 μl of 0.05 M NaOH were added to thecell pellets. Samples were boiled for 10 min., then 6 μl of 1 M Tris (pH7.2) were added. Samples were then centrifuged at 12,000×g andsupernatants were used for assay. The 5′- and 3′-oligonucleotide primerschosen for amplification and the PCR reaction conditions are describedin Pugatsch et al. Reaction products were visualized on 1.6% agarosegels (Sigma, USA) containing 0.05 μg/ml ethidium bromide.

Murine BCL1

BCL1, a B-cell leukemia/lymphoma of BALB/c origin (Slavin, S. andStrober, S. Nature, 272:624 (1978); Slavin, S. et al. Cancer Res.,41:4162 (1981)), was maintained in vivo by serial passage in BALB/cmice. Inoculation of 10 to 100 BCL1 cells in BALB/c mice results intypical B-cell leukemia/lymphoma characterized by splenomegaly, extremeperipheral blood lymphocytosis (up to 500,000 lymphocytes/ml) and deathof 100% of recipients. BCL1 also causes leukemia in F1 recipients, butdevelopment takes longer than in BALB/c recipients (Slavin et al.(1981), supra).

Immunization of Donor Mice

Donor C57BL/6 (H2-^(b)) mice were immunized against alloantigens of boththe MHC and Minor Histocompatibility Loci (MiHL) by injection witheither spleen cells obtained from BCL1-bearing BALB/c (H-2^(d)) mice(30×10⁶ cells per mouse per immunization) or spleen cells obtained fromnormal BALB/c mice (30×10⁶ per mouse). Donor B10.D2 (H-2^(d)) mice wereimmunized against MiHL alloantigens only by injection with the same cellpopulations used for immunization of the C57BL/6 mice. Immunizationswere by intraperitoneal injection on days −20 and −10 prior tosacrifice, harvesting of spleens, and transfer of isolated spleen cellsto F1 or BALB/c host animals.

Total Body Irradiation (TBI)

Mice were conditioned by TBI with a single dose of 400 cGy delivered bylinear accelerator (Varian Climac 6×) at a source to skin distance of 80cm, at a dose rate of 107 cGy/min.

Transplantation of Spleen Cells for Immunotherapy of BCL1

Spleens from C57BL/6 or B10.D2 donors immunized using the protocolsdescribed above were teased into single cell suspensions using nylonmeshes, washed twice with 10% bovine calf serum in RPMI 1640 medium(Biological Industries, Beit Haemek, Israel), and injectedintravenously.

GVL Effects Against BCL1

Assays for assessing induction of GVL were performed as follows. Fortesting GVL across an incompatibility involving both MHC and MiHLalloantigens, total of 10⁴ fresh BCL1 cells and 30×10⁶ spleen cellsobtained from the immunized or control unimmunized C57BL/6 donors wereinfused into F1 recipients 24 h after TBI. For testing GVL across anincompatibility involving only MiHL alloantigens, a total of 2×10³ BCL1and 30×10⁶ spleen cells obtained from immunized or control unimmunizedB10.D2 donors were infused into BALB/c recipients 24 h after TBI.Administration of a known tumor cell number allowed quantitativemeasurement of GVL effects.

Assay for Chimerism

Chimerization of BALB/c or F1 recipients by C57BL/6 spleen cells wasmeasured by testing the percentage of host or donor-type cells in spleenor blood samples using an in vitro complement-dependentmicrocytotoxicity assay with specific alloantisera and rabbitcomplement. Morecki et al. J. Exp. Med., 165:1468 (1987). Specificalloantisera (“BALB/c anti-C57BL/6” and “C57BL/6 anti-BALB/c”) wereprepared by cross-immunization of the relevant mice with full-thicknessskin allografts followed by 6 intraperitoneal injections of 30×10⁶ donorspleen cells at intervals of 1-2 weeks. Mice were bled and sera storedat −70° C.

Spleen or peripheral blood cells from the BALB/c or F1 recipients wereincubated with both alloantisera and rabbit complement. Host BALB/ccells were lysed with anti-BALB/c and not anti-C57BL/6 antisera, host F1cells were lysed with both anti-BALB/c and anti-C57BL/6 antisera, andcells in the BALB/c F1 mice derived from the C57BL/6 donor mice werelysed by only anti-C57BL/6 antiserum. Chimerism was expressed as % donortype (C57BL/6) cells which was calculated as follows:

% donor type (C57BL/6) cells=% cells lysed with anti-C57BL/6 antiserum-%cells lysed with anti-BALB/c antiserum-% cells lysed with complementalone.

Serial PBL Counts for Monitoring Development of BCL1-Leukemia

Peripheral blood samples (20 μl) were obtained by weekly venipunctureusing heparinized glass capillaries. Peripheral blood leukocyte (PBL)counts were determined using a hemocytometer after lysis of red bloodcells in 2% acetic acid.

Detection of Residual BCL1 Cells by Adoptive Transfer

An in vivo assay was used for detection of residual BCL1 cells intreated experimental mice. Aliquots of 10⁵ lymphocytes obtained from apool of spleen cells from treated mice with no evidence of disease wereadoptively transferred to normal secondary BALB/c recipients (10 miceper group). The development of leukemia was determined by weeklyperipheral blood lymphocyte (PBL) counts, monitoring spleen enlargement,and survival.

Monitoring of GVHD

Recipients were monitored for survival and clinical signs of GVHD suchas ruffled fur, diarrhea, and measurable weight loss. At the time ofadoptive transfer, samples of liver and lung sections were obtained formeach mouse to test for histological evidence of GVHD. Histologicalpreparations were analyzed by an independent pathologist on a doubleblind basis.

Cytokine Assays

The levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α),interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10)were measured in supernatants of donor (C57BL/6) spleen cells at thetime of harvesting for transplantation to host (recipient) animals.Recipient (F1) spleen cell supernatant cytokine levels were measured at3 weeks after transplantation of donor C57BL/6 spleen cells. Cytokineswere measured by “sandwich” ELISAs using murine cytokine-specificmonoclonal antibodies for capture using standard methods familiar tothose in the art. Antibodies that bind to cytokine determinants that donot overlap with determinants that bind the capture antibodies andalkaline phosphatase conjugated anti-Ig antibodies were used fordetection. Standard curves were generated with standard samples of eachcytokine and the concentrations of the cytokines in unknown samples weredetermined from the standard curves.

In vitro T Cell Proliferation Assays

Mitogen-induced T cell proliferative responses were measured byculturing spleen cells in the presence of the T cell mitogensconcanavalin A (ConA, 10 μg/ml) or phytohemagglutinin (PHA, 1 μg/ml) forthree days. [³H]-thymidine was added to the cultures for the last 6hours of culture and T cell proliferation was measured in terms ofcounts per minute (cpm) of [³H]-thymidine incorporated into the DNA ofthe cells using cell harvesting and radioactivity techniques known tothose in the art.

In vitro Cell-mediated Lysis Assays

Human peripheral blood mononuclear cell (PBMC) and murine spleen or bonemarrow populations were tested for their ability to lyse NK-sensitivetarget cells (human K562 and murine YAC-1 cells) and NK-resistant,activated NK-sensitive target cells (human Daudi and murine P815 cells)using standard ⁵¹Cr-release assays. Briefly, ⁵¹Cr-labeled target cellswere incubated at 37° C. with PBMC effector cells at various target cellto effector cell ratios for 4 hours. At the end of the incubation, thecells were pelleted by centrifugation and equal aliquots of supernatantwere removed from each assay culture well and counted for radioactivity.Percent lysis was calculated by the following formula:${{Percent}\quad {lysis}} = {\frac{{{cpm}\quad \left( {\exp.} \right)} - {{cpm}\quad \left( {{cont}.} \right)}}{{{cpm}\quad \left( {\max.} \right)} - {{cpm}\quad \left( {{cont}.} \right)}} \times 100}$

where:

cpm(exp.) are the cpm detected in the supernatant from an experimentalculture well containing target cells and effector cells;

cpm(cont.) is the mean value of the cpm detected in supernatants fromcontrol culture wells containing target cells but no effector cells; and

cpm(max.) is the mean value of the cpm detected in supernatants fromculture wells containing target cells and a detergent (e.g., sodiumdodecyl sulfate) or acid (e.g., 1N HCl) and represent the maximum amountor radioactivity releasable from the target cells by lysis.

Statistical Analyses

The statistical significance of the results comparing treated andcontrol mice was calculated by the independent t-test.

EXAMPLE 2 Nonspecific Immunosuppression of Mice Treated with sTLI Aloneor with sTLI and Cy

In the first set of experiments (Table 1), BALB/c mice were given 6, 8or 12 (experimental groups) or 17 (control groups) fractions of TLI at200 cGy/fraction. After TLI, 3×10⁷ BMC from B6 donors were administeredone day after the last TLI. Skin allografts from B6 donors weretransplanted 20 days after transfer of the BMC.

In a second set of experiments (Table 2), 5 groups of BALB/c recipientswere administered sTLI of 6 fractions of 200 cGy/day, followed a daylater with 200 mg/kg Cy intraperitoneally. After the above conditioning,the five groups were treated as described below. Group 1 received 3×10⁷BMC, whereas group 2 received 3×10⁷ BMC and 5×10⁶ spleen cells. Group 3received 0.3 ml of whole blood. Group 4 received 3×10⁶ BMC, whereasgroup 5 received 3×10⁶ T cell depleted BMC. T cell depletion wasperformed in vitro with monoclonal antibody Thy 1 and rabbit complement,as described in Example 1. For all of the above five groups, cells andwhole blood were from B6 donors and were infused intravenously. Skinallografts were transplanted 20 days after BMC or blood transfer.

Results

A short course of TLI (sTLI), in contrast to a long course of TLI (17fractions, 200 cGy each), was insufficient for acceptance of stem cellsfrom allogeneic BMC or blood. Table 1 shows that none of the BALB/c micereceiving 3×10⁷ fully mismatched BMC from B6 donors became hematopoieticcell chimeras after 6 fractions of TLI, while consistent acceptance ofallogeneic BMC was obtained after 17 fractions of TLI. As shown in Table1, after treatment with sTLI and allogeneic BMC or allogeneic bloodcells, BALB/c recipients stayed alive, none developed GVHD and allrejected B6 skin allografts. Thus, after sTLI alone, sufficient numbersof immunocompetent cells remain in the host to reject a donor allograft.

TABLE 1 Incidence of allogeneic BMC and skin graft acceptance afterfractionated total lymphoid irradiation. No of % of donor cells Skinallograft TLI in blood 100 days survival Fractions after celltransfer^(a) >100 days^(c)  6 0 (10)^(b)  0/10  8 0 (3) , 56 (1) 1/4 120 (3) , 50, 90 2/5 17 82, 85, 90 (2), 93 5/5 ^(a)BMC (3 × 10⁷) from B6donors were given to BALB/c recipients one day after the last TLI.^(b)Number of mice with the same level of chimerism is given inparentheses. ^(c)Skin allografts from B6 donor were transplanted 20 daysafter cell transfer.

TABLE 2 Skin allograft survival and GVHD related death aftertransplantation of allogeneic cells and skin graft following sTLI incombination with a single injection of Cy. Mice Skin Donor GVHD survivalgraft Cells^(a) related Mean ± SD survival^(c) No day 0 death days >100days 1 BM 3 × 10⁷ 22/24 39 ± 8 2/2 2 BM 3 × 10⁷ 14/14 10 ± 3  NT andspleen - 5 × 10⁶ 3 Blood 7/7 30 ± 12 NT 0.3 ml 4 BM 3 × 10⁶ 14/15 40 ±7  1/1 5 BM 3 × 10⁶ 0/7 >100 1/7 T cell depleted^(b) ^(a)Cells or wholeblood from B6 donors were transferred intravenously to BALB/c recipientsafter sTLI and Cy. ^(b)T cell depletion was performed in vitro with mAbanti Thy 1 and rabbit complement. ^(c)Donor skin allografts weretransplanted 20 days after cell transfer. NT - not tested.

In sharp contrast, a single injection of Cy (200 mg/kg) given one dayafter the last fraction of sTLI (6 fractions, 200 cGy each), increasedthe non-specific immunosuppression and allowed the hosts to accept BMC,spleen and blood cells. However, all recipients developed typical acuteGVHD which was lethal in most cases (Table 2). The survival time of therecipients with GVHD appeared to be a function of the number of matureimmunocompetent T cells present in the inoculum. Mean survival time ofmice inoculated with 3×10⁷ BMC was four times longer as compared withrecipients of an equal number of BMC mixed with 5×10⁶ B6 spleen cells(Table 2, groups 1 & 2). Transfer of fewer BMC (3×10⁶ instead of 3×10⁷)did not prolong survival significantly (Table 2, groups 1 & 4).

GVHD was successfully prevented in the mice of group 5 (Table 2), whoreceived T cell depleted BMC after sTLI and Cy. Although none of theseexperimental mice developed GVHD and all of them remained alive, all butone rejected donor BMC. Accordingly, prolonged skin allograft survivalwas observed in only 1/7 recipients.

EXAMPLE 3 Antigen-specific Elimination of Residual Donor-alloreactiveHost Immunocompetent T Cells

Three groups of BALB/c recipient mice were administered sTLI in 6fractions of 200 cGy/day. Non T cell depleted BMC (day 0) from B6 donorswere transferred intravenously to the BALB/c recipients in all threegroups. The next day all of the recipients were administered 200 mg/kgof Cy. One group (Table 3, Group 1) received a skin graft from B6 donorsat day 20. The second group (Table 3, Group 2) received 3×10⁷ non T celldepleted BMC from B6 donors on day 2 followed by a skin graft at day 20.The third group (Table 3, Group 3) received 3×10⁶ non T cell depletedBMC from B6 donors on day 2 followed by a skin graft at day 20.

Levels of donor cells in blood were assayed in all surviving mice at day100 according to the protocol of Example 1.

TABLE 3 BM and skin allograft acceptance in experimental mice. Treatmentof Mice after TLI × 6 Skin GVHD Survival Donor Cy Donor Related in MiceBMC^(a) 200 Donor Skin Cells in Death Without 3 × 10⁷ mg/kg BMC GraftingBlood days GVHD No day 0 day 1 day 2 day 20 day 100 means ± SD^(e)days >100 1 + + — + <20% (8)^(d), 2/10 2/8 20%, 37% 75,85 2 + + 3 ×10⁷ + NT 15/15 — 35 ± 5 3 + + 3 × 10⁶ + NT 20/22 2/2 67 ± 12 >130, >2804 + + 3 × 10⁶ + 20-50% 0/6 6/6 T Cell 132, >270(5) Depleted in Vitro^(b)5 + + 3 × 10⁶ + 20%-50% 10/20 10/10 T Cell 48 ± 10 >230(10) Depleted inVivo^(c) ^(a)BM cells from B6 donors were transferred intravenously toBALB/c recipients. ^(b)T cell depletion was performed in vitro with mAbYTS 148.3 and rabbit complement. A dose of 2 × 10⁶ cells/recipientresulted in similar tolerance (data not shown). ^(c)T cell depletion wasperformed in vivo with mAb YTS 154.7. ^(d)Number of mice is given inparentheses. ^(e)Mean ± SD survival time of mice. NT - not tested.

Results

Most of the mice converted to mixed chimeras with a relatively lownumber (7%-20%) of donor hematopoietic cells in the blood (Table 3,group 1). Donor non T cell depleted BMC transplanted one day after theCy engrafted successfully but induced GVHD (Table 3, groups 2, 3).

EXAMPLE 4 Establishment of Stable GVHD-free Mixed Chimeras by Transferof Low Dose T Cell Depleted Donor BMC

Two groups of BALB/c recipient mice were treated as described in Example3 except that T cell depleted BMC were administered on day 2 instead ofnon T cell depleted BMC. One group (Table 3, Group 4) was administered3×10⁶ of in vitro T cell depleted BMC from B6 donors. A total of 2×10⁶ Tcell-depleted BMC was sufficient, as demonstrated in one additionalexperiment (data not shown). The other group (Table 3, Group 5) wasadministered 3×10⁶ of in vivo T cell depleted BMC from B6 donors. T celldepletion was performed as described in Example 1.

Results

Elimination of immunocompetent T cells from allogeneic donor BMC wascrucial for prevention of GVHD (FIG. 1). In mildly immunosuppressedrecipients, after in vitro depletion of T cells from donor BMC, alltreated mice converted to stable mixed chimeras with 20%-50% of donorcells in the blood. The stable mixed chimeric mice acceptedfull-thickness B6 skin allografts and survived for 152-290 days withoutclinical signs of GVHD (Table 3, group 4). Similar results were obtainedusing an identical protocol for BALB/c→B6 chimeras with permanent (>150days) survival of BALB/c skin allografts (data not shown).

Depletion of T cells in vivo from donor BMC was less successful than Tcell depletion in vitro (Table 3, group 5). Of the mice that received invivo T cell depleted BMC, only half remained free of GVHD and survivedfor >250 days (Table 3, group 5). These animals were all confirmed to bestable mixed chimeras and all accepted donor skin allografts.

EXAMPLE 5 Tolerance to Allografts of Donor BM Stroma and Neonatal Heart

Five groups of BALB/c recipient mice were conditioned with sTLI byadministration of 6 fractions of 200 cGy/day. All of these groups thenreceived 3×10⁷ non T cell depleted BMC of B6 donors intravenously oneday after the last TLI fraction. A dose of Cy (200 mg/kg) was given oneday after the BMC transfer but before allograft transplantation.Twenty-four hours after the Cy, Group 1 (Table 4) was transplanted withnon T cell depleted BMC whereas Group 2 (Table 4) was transplanted within vitro T cell depleted BMC. Group 3 (Table 4) was transplanted withBMC stroma one day after Cy, Group 4 (Table 4) with heart 20 days afterCy and Group 5 with skin 20 days after Cy. All of the allografts werefrom B6 donors.

TABLE 4 Acceptance of various cell and tissue allografts in hostsconditioned with STLI, donor-derived bone marrow cells and Cytoxan. %Donor Cells in Blood Before Graft Allograft Survival Group Trans- Typeof Allograft Time in Number^(a) plantation Allograft Acceptance days^(h)1 <20 BMC 22/22^(g) 17-25 (20)^(h) >150 (2) 2 <20 BMC, T cell  6/6152, >290 (5) depleted 3 <20 BM stroma^(c)  8/8^(e) >220 (8) 4 <20Heart^(d)  5/6^(f) 80, >155 (4) 5 <20 Skin^(d)  0/13 6 20-50 Skin^(d)13/14 >270 (13) In vitro T cell depleted BMC^(b) 1 day after Cy^(a)Recipients in groups 1-5 were conditioned prior to transplantationwith sTLI (6 daily exposures of 200 cGy), 3 × 10⁷ BMC intravenously oneday after the last TLI fraction and 200 mg/kg Cy intraperitoneally oneday after cell transfer. ^(b)One group of recipients (group 6) wereinoculated with a second graft consisting of 3 ×10^(6 T cell depleted (in vitro) BMC from B6 donors given one day after Cy.)^(c)BM plugs were grafted on day after Cy. ^(d)Heart muscle or skinallografts were grafted 20 days after Cy. ^(e)Ectopic bone formationunder the kidney capsule was confirmed by X-ray analysis. ^(f)Viabilityand regular pulsatile activity of heart muscle allografts was confirmedby ECG. ^(g)Twenty recipients in group 1 died from GVHD 37-45 days aftercell transfer. ^(h)Number of mice surviving with allografts is indicatedin parentheses.

Results

Mice that were given a second infusion of unmanipulated donor BMC hadgraft acceptance but 20 of the 22 mice died from GVHD 37-45 days aftercell transfer. Mice transplanted with T cell depleted BMC in the secondinfusion had graft acceptance and much higher graft survival.

Implantation of two femoral plugs from B6 donors under the kidneycapsule of BALB/c recipients one day after Cy without a second inoculumof T cell depleted donor BMC resulted in formation of fully developedectopic bone confirmed by X-ray analysis and subsequently by autopsy.This bone supported both donor and recipient hematopoiesis (Table 4,Group 3). Fragments of the ectopic osteo-hematopoietic site, whenretransplanted under the kidney capsule of normal mice, formed bones andectopic hematopoietic sites in secondary recipients of donor origin (9/9successful allografts in B6 recipients) but not in BALB/c mice (0/9).These data indicate that ectopic osteo-hematopoietic sites in these micewere of donor origin.

The same treatment was also sufficient for acceptance of heterotopicallytransplanted neonatal heart grafts obtained from 1-2 day old B6 donors.Results show that the heart muscle transplanted into an ear skin pocketof BALB/c recipients 20 days after the tolerogenic treatment were ECGpositive for >80 days (Table 4, Group 4). In all mice that accepteddonor-derived neonatal heart grafts, contractions of the heart musclecould also be detected visually. Mice that received only sTLI and Cyrejected both femoral plugs and neonatal heart grafts from B6 donorswithin 30 days (Data not shown).

Most of the recipients that received sTLI, BMC and Cy accepted donorBMC, BM stromal precursor cells and neonatal heart allografts. However,the conditioning was not sufficient to ensure survival of skinallografts obtained from the same donor (Tables 3 & 4).

EXAMPLE 6 Conditions Required for Stable Donor Skin Allograft Acceptancein Mice

A group of mice were administered sTLI, BMC and Cy as described inExample 5. This group (Table 4, Group 6) also received a secondinoculation of 3×10⁶ of B6 in vitro T cell depleted B6 donor BMC one dayafter Cy but prior to skin allograft.

Results

Mixed hematopoietic cell chimerism was documented among all experimentalanimals tested, those that accepted as well as those that rejecteddonor-type skin allograft. However, the level of chimerism was clearlyhigher in the mice that, after administration of Cy, received donor BMCin suspension or within a BM femoral plug (20%-50% donor cells) ascompared with mice that received no second infusion with BMC (<20% donorcells). These data indicate that skin allograft acceptance, which is astrong immunogen, is dependent on the level of hematopoietic cellchimerism in recipients.

Mixed chimeras with 20% or more donor cells in their blood accepteddonor skin allografts for >270 days without any additional treatment(Table 4, group 6). Most of the mice that did not receive the secondinoculum of donor BMC had less than 20% donor cells in the blood andrejected donor skin allografts (Table 4, group 5). This same group ofmice nonetheless accepted other donor-derived tissues. These datademonstrate that although a relatively low number of donor cells in theblood (e.g., less than about 20%) may be sufficient for successfulengraftment of marrow-derived stromal cells and heart grafts, consistentacceptance of skin allografts derived from the same donor across strongMHC barriers requires a higher level of hematopoietic cell chimerism.

EXAMPLE 7 Specificity of Transplantation Tolerance Induced by theTolerogenic Treatment

Tolerant BALB/c recipients with intact B6 skin allografts for 150 daysrejected (11/11), within 18-20 days, a second skin allograft obtainedfrom a third party (CBA) donor while keeping intact the original B6 skinallograft. This indicates that donor-type specific transplantationtolerance was induced and maintained in recipients capable of generatingnormal immune responses with full expression of alloreactivity tonon-relevant transplantation antigens.

Acceptance of donor skin allografts was observed in all straincombinations investigated including, DBA→BALB/c (n=10), B6→CBA (n=3),B6→BALB/c (n=21) and BALB/c→B6 (n=9).

EXAMPLE 8 Application of Tolerogenic Treatment for Induction ofTransplantation Tolerance to Skin Xenografts in Rat→Mouse Combination

Two groups of mice were administered sTLI of 6 fractions of 200 cGy/day.After sTLI conditioning, both groups were administered 30×10⁶ non-T celldepleted Lewis rat BMC intravenously. The first group was given a singledose of 200 mg/kg Cy the next day. Another 3×10⁷ non T cell depleted ratBMC were administered the following day. In the second group, a dose of60 mg/kg Cy was given daily for 3 days in contrast to the single 200mg/kg dose given the first group. The first dose of Cy was given 10hours after the first rat BMC inoculation, the second dose at 24 hoursand the third dose at 48 hours (see below). After administration ofthree doses of 60 mg/kg Cy, a second inoculation of 3×10⁷ non T celldepleted rat BMC were administered.

Results

The first group of mice treated as described above (Table 5, group 1)accepted the second inoculum of 3×10⁷ non T cell depleted BMC from Lewisrats. Lethal GVHD was induced, however, in most of the recipients,suggesting fast engraftment of donor cells despite the relatively mildand non-myeloablative immunosuppressive conditioning (Table 5, group 1).Interestingly, mice that developed GVHD, indicating acceptance of donorcells, were still capable of rejecting donor-derived skin grafts priorto succumbing to the disease. These results confirm the observation thatresidual donor-reactive host T cells mediating host vs graft reactionmay survive the immunosuppressive/tolerogenic treatment and causerejection of highly immunogenic donor-derived skin xenografts.

TABLE 5 Tolerance in mice to Lewis rat bone marrow cells and skinxenografts. Treatment of Mice After sTLI Conditioning Skin XenograftSkin Survival Graft Time Day 0 Day 1 Day 2 Day 3 Acceptance in days^(d)Rat Cy Rat  0/6 <20(6) BMC^(a) 200 BMC^(c) mg/kg Rat Cy^(b) Cy Cy Rat15/20 >176(2), BMC^(a) 60  60 60 BMC^(c) 123, mg/kg mg/kgmg/kg >95(5) >67(3), 39(2), 24(2)^(e) ^(a)3 × 10⁷ Lewis BMCintravenously. ^(b)1st injection with Cy 10 h after the 1st rat BMCinoculation. ^(c)3 × 10⁷ Lewis BMC intravenously. ^(d)In parenthesesnumber of mice keeping rat skin graft for indicated period. ^(e)5/8 micedied from GVHD with rat skin xenograft accepted. Seven of 15 mice withintact skin allografts developed no acute GVHD.

The results improved, in the second group, when Cy was divided intothree equal doses of 60 mg/kg and injected 10 h, 24 h and 48 h after thefirst infusion of non-T cell depleted rat BMC. Under these conditions,15 of 20 B6 recipients accepted full thickness Lewis rat skin xenografts(Table 2, group 2). By modifying the Cy administration protocol, hostxenoreactive cells may have been more effectively controlled. Normaldonor hair growth was observed in all 15 recipients, 5 of whichdeveloped lethal GVHD. The surviving mice did not develop clinical signsof GVHD although they were transplanted with non T cell depletedxenogeneic BMC suggesting that residual host-type hematopoietic cellsmay down-regulate donor-derived immunocompetent T cells through a vetoeffect.

EXAMPLE 9 Effect of Varying Doses of TLI on GVHD-free and Donor-typeSkin Allograft Survival

BALB/c mice were treated with 0 to 6 doses of sTLI, with each dose being200 cGy. After administration of sTLI, one group of mice received Cy(200 mg/kg), followed, one day later, by 3×10⁷ or 3×10⁶ BMC (non-Tcell-depleted) obtained from B6 donors. A second group of mice received,one day after sTLI, 3×10⁷ BMC (non-T cell-depleted) from B6 donors.These mice, 24 hours later, were administered Cy (200 mg/kg). One dayafter the Cy, these mice again received 3×10⁷ or 3×10⁶ non-Tcell-depleted BMC also obtained from B6 donors. A third group of micereceived, after sTLI, 0.3 ml of blood from B6 donors and, 24 hourslater, Cy (200 mg/kg). Once again, one day after Cy, 3×10⁷ or 3×10⁶non-T cell-depleted BMC, obtained from B6 donors, were administered tothese mice. Twenty days later, donor B6 skin was grafted into survivingmice of all the groups.

Another set of experiments was conducted to correlate donor cell levelsand skin allograft acceptance. The tolerogenic treatment includedvarying numbers of sTLI doses, followed, a day later, by 3×10⁷ BMC and,24 hours later, Cy (200 mg/kg). A day after administration of Cy, asecond infusion of 3×10⁷ BMC was administered. Donor skin was grafted 20days later. Percentages of donor blood cells in host mice were evaluatedat 100-130 days after skin grafting.

In another set of experiments, sTLI-treated BALB/c mice were tolerizedas indicated in Table 6 followed, 20 days later, with donor skinallografts. At either day 100 or day 120, donor cell chimerism wasassayed and mixed lymphocyte reaction (MLR) tests were performed. The Tcells were then enriched by lysing red blood cells with ammoniumchloride, followed by passage through a nylon wool column andreconstituting in RPMI medium supplemented with 10% of AB Human serum,0.09 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine,100 μg/ml streptomycin and 100 U/ml penicillin (Biological Industries,Beit Haemek, Israel) and 0.05 mM 3-mercaptoethanol (Sigma, USA). 10⁵responding T cells were incubated with 10⁶ stimulating T cells (3000 cGyirradiated) in flat bottom microplates (Costar, USA) at 37° C., 5% CO₂for 3 days. The cells were pulsed on the third day with 1 μCi [³H]thymidine and harvested on the fourth day.

TABLE 6 MLR reactivity of BALB/c mice that accepted or rejecteddonor-type skin allografts. Treatment MLR with Stimulators of Time DonorIrradiated 3000 cGy Mice After Acceptance Cells (cpm × 10⁻³) TreatedTreatment of B6 Skin in Auto- Mice in MLR^(a) (Days) Allograft^(b)Blood^(c) Responders logous B6 BALB/c 1 BMC, Cy, 100 + 65% 1 2.24 1.932.00 3 × 10⁷ BMC 2 BMC, Cy, 100 + 73% 2 1.72 1.56 2.89 3 × 10⁷ BMC 3BMC, Cy, 100 + 56% 3 1.36 2.45 3.29 3 × 10⁷ BMC 4 BMC, Cy, 120 —  7% 45.80 35.45 6.56 3 × 10⁶ BMC 5 BMC, Cy, 120 — 12% 5 11.98 58.08 16.07 3 ×10⁶ BMC 6 BMC, Cy, 120 —  7% 6 14.64 28.34 11.94 3 × 10⁶ BMC 7 BMC, Cy,120 —  7% 7 21.15 66.17 29.66 3 × 10⁶ BMC ^(a)BALB/c mice that received3 × 10⁷ BMC from B6 donors and 24 h later, 200 mg/kg Cy werereconstituted with 3 × 10⁷ BMC (mice 1-3) or with 3 × 10⁶ BMC (mice 4-7)from B6 donors one day after injection of Cy. ^(b)Skin allografts fromB6 mice were transplanted 20 days after treatment. ^(c)Percentage ofdonor cell chimerism was assayed on the same day as MLR.

Results

In the absence of donor-specific tolerization (FIG. 2A, Group A), 0, 1,or 2 doses of sTLI appeared to be sufficient for a high probability ofGVHD-free survival when 3×10⁷ BMC or 3×10⁶ BMC were administeredsubsequent to the Cy treatment. Administration of these low doses ofsTLI to a host results in retention of relatively high numbers of hostfunctional T cells. Consequently, the relatively high rate of GVHD-freesurvival at low sTLI doses may be due to a veto effect in which thelevels of host- and donor-derived veto cells are in balancedequilibrium. Donor skin graft acceptance was low at 0 and 1 sTLI dosebut increased at 2, 3, and 6 sTLI doses (data not available for 4 and 5sTLI fractions). However, the GVHD-free survival rate decreased at thesedoses of sTLI. (FIG. 2A). Thus, in the absence of donor-specifictolerization, none of the regimens led to a high percentage of GVHD-freesurvival and a high percentage of donor skin graft acceptance.

Donor-specific tolerization in the second group of mice (FIG. 2A, GroupB) resulted in high GVHD-free survival at low doses of sTLI, and evenwith no TLI treatment. The donor-specific tolerization also resulted ina high probability of donor skin allograft acceptance regardless of thenumber of sTLI doses. Without any sTLI fractions, a second dose of 3×10⁷BMC appears to be necessary. The use of higher numbers of sTLI fractionsresulted in higher GVHD morbidity, although skin allograft acceptanceremained high. With regards to GVHD, the higher numbers of sTLIfractions seem to be more successful with a lower dose (3×10⁶) of BMC.This may be due to a veto effect in which lower numbers of host vetocells, due to administration of higher numbers of sTLI fractions, are inbalanced equilibrium with a lower dose of BMC.

The infusion of 0.3 ml of blood, without any TLI, in the third group ofmice (FIG. 2C, group C) resulted in a high probability of GVHD-freesurvival but low donor skin allograft acceptance. This third group ofmice had high GVHD-free survival when 0, 1, or 2 fractions of sTLI wereadministered and the mice received a large dose of (3×10⁷) BMC followingCy. However, higher numbers of sTLI fractions were more successful witha smaller dose of (3×10⁶) BMC following Cy. These results may be due toa veto effect as discussed above for the second group of mice.

The correlation between the percentages of donor blood cells in arecipient and donor-skin allograft acceptance is illustrated in FIG. 3.These experiments indicated that the percentage of donor blood cells inthe recipient was critical for skin allograft acceptance. Recipientswith less than 20-25% donor blood cells did not accept donor-skinallografts (solid symbols). In contrast, recipients having greater than20-25% donor blood cells accepted donor-skin allografts (empty symbols).Furthermore, recipients were able to obtain greater that 20-25% of donorblood cells even when conditioned with 0, 1, 2, or 3 sTLI fractions inthe tolerogenic treatment.

MLR reactivity data (Table 6) indicated that mice with low levels ofchimerism (mice 4-7) were not completely tolerized. The responder Tlymphocytes proliferated in the presence of stimulators from autologous,BALB/c and B6 sources. The response to B6 stimulators was especiallyhigh. In contrast, mice with high levels of chimerism (mice 1-3) did notrespond to stimulators from any of the sources.

EXAMPLE 10 Immunized Donor Spleen Cells Induce Stronger GVL EffectsAcross an MHC Barrier than Unimmunized Spleen Cells

In order to measure GVL activity across an incompatibility involvingboth MHC and MiHL alloantigens, F1 mice (pre-conditioned on day 0 withTBI (400 cGY)) were injected on day 1 with 10⁴ BCL1 cells and 30×10⁶immunized or normal C57BL/6 spleen cells.

There were 4 test groups with 10 mice in each:

Group 1: F1 recipients given BCL1 cells and spleen cells from C57BL/6mice immunized with spleen cells from overtly leukemic BCL1-bearingmice.

Group 2: F1 recipients given BCL1 cells and spleen cells from C57BL/6mice immunized with normal BALB/c spleen cells.

Group 3: F1 recipients given BCL1 cells and normal C57BL/6 spleen cells.

Group 4: Untreated F1 recipients given BCL1 cells only and therebyserving as a control group.

Results

FIG. 4 shows cumulative data from 4 similar experiments. 75% of the micein the 1st group and 48% of the mice in the 2nd group were alive after120 days with no evidence of leukemia. The data described in thefollowing paragraph and shown in FIG. 5 indicate that the 25% of mice ingroup 1 that died before 120 days, died of GVHD rather than leukemia. Incontrast, 79% of the mice in the 3rd group died of GVHD within 75(median 56) days. All mice in group 4 died of leukemia within 36 (median24) days. The therapeutic advantage of BCL1-immunized C57BL/6 spleencells over BALB/c spleen cell-immunized C57BL/6 spleen cells andBCL1-immunized spleen cells over normal, unimmunized C57BL/6 spleencells was significant (p=0.001 and 0.002 respectively). Although F1 micegiven spleen cells from BALB/c spleen cell-immunized C57BL/6 miceapparently had a higher survival rate compared with F1 mice treated withnormal, unimmunized C57BL/6 spleen cells (FIG. 4), the advantage was notsignificant (p=0.083).

To measure the efficacy of allogeneic cell therapy in eradicatingresidual BCL1 cells, spleen cells obtained 3 weeks post transplantationwere pooled from each experimental group and 10⁵ cells were adoptivelytransferred to secondary BALB/c recipients (10 mice/group) (FIG. 5). Theresults were consistent with those obtained in the primary F1 recipientmice. The GVL effect of BCL1-immunized spleen cells was more potent thanthat of both BALB/c spleen cell-immunized C57BL/6 spleen cells (p=0.001)and normal, unimmunized C57BL/6 spleen cells (p=0.004). The BALB/cspleen cell-immunized C57BL/6 spleen cells showed no greater anti-tumoractivity than normal, unimmunized C57BL/6 spleen cells (p=0.5). All thesecondary recipients inoculated with spleen cells obtained from the 1stexperimental group remained leukemia-free for >120 days. 25 of 40secondary BALB/c recipients inoculated with spleen cells obtained fromthe 2nd experimental group remained leukemia-free for >120 days, and 27of 40 secondary BALB/c recipients inoculated with spleen cells obtainedfrom the 3rd experimental group remained leukemia free for >150 days. Incontrast, all 28 secondary BALB/c recipients of spleen cells obtainedfrom the control group died of leukemia within less than 30 days. PBLtaken from F1 recipients showed 46-56% donor-type cells, confirmingengraftment (Table 7).

TABLE 7 Chimerism in F1 recipients transplanted with C57BL/6 spleencells. Donor Type F1 Mice Injected with Spleen Cells From Cells (%)C57BL/6 mice immunized with BCL1 50 ± 12 C57BL/6 mice immunized withBALB/c spleen cells 46 ± 14 Normal C57BL/6 mice 56 ± 16

Body weight studies of the mice in each group, as an objective measureof the degree of GVHD, are shown in FIG. 6. All mice had severe GVHD inthe first 2 weeks after transplantation and a proportion of the animalsin each group did not survive. However, 75% of the mice in the 1st groupand 48% of the 2nd group remained active and well with no evidence ofGVHD at 120 days. All animals in the 3rd group died within 75 (median56) days with typical signs of acute GVHD. F1 mice that were givennormal C57BL/6 spleen cells showed typical clinical signs of GVHDmanifested by a continuous weight loss, whereas transient reduction inbody weight for only one week was observed for F1 mice receiving C57BL/6spleen cells obtained from donors immunized against either BCL1 ornormal BALB/c spleen cells.

EXAMPLE 11 Immunized Donor Spleen Cells Induce Stronger GVL EffectsAcross an MiHL Barrier than Unimmunized Spleen Cells

In order to determine the GVL effects across MiHL incompatibility,BALB/c recipients (conditioned on day 0 with TBI (400 cGy)), wereinjected intravenously on day 1 with 2×10³ BCL1 cells and 30×10⁶ spleencells from either B10.D2 mice pretreated in one of three ways or noB10.D2 spleen cells. There were 4 experimental groups with 10 mice ineach:

Group 1: BALB/c recipients given BCL1 cells and spleen cells from B10.D2immunized with BCL1 cells obtained from BALB/c.

Group 2: BALB/c recipients given BCL1 cells and spleen cells from B10.D2mice immunized with normal BALB/c spleen cells.

Group 3: BALB/c recipients given BCL1 cells and normal B10.D2 spleencells.

Group 4: Untreated BALB/c recipients given BCL1 cells only and therebyserving as a control group.

Results

As summarized in FIG. 7, 84% of the mice in the 1st group were alivewith neither signs of clinical GVHD nor leukemia for >120 days.Similarly, 47% of the mice in the 2nd group were alive with no sign ofGVHD or leukemia for >120 days, whereas 16 of 30 died of leukemia. Allmice in the third and fourth groups died of leukemia. There was asignificant therapeutic advantage of treating the recipient BALB/c micewith spleen cells from BCL1-immunized B10.D2 mice over treatment witheither spleen cells of BALB/c spleen cell-immunized B10.D2 mice (p=0.03)or spleen cells from naive, unimmunized B10.D2 mice (p<0.002) (FIG. 7).There was also a significant therapeutic advantage in administeringspleen cells from BALB/c spleen cell-immunized B10.D2 animals overadministering spleen cells from naive, unimmunized B10.D2 mice(p=0.0001). Spleen cells from BCL1-immunized B10.D2 mice displayedgreater GVL-mediated therapeutic efficacy than did those from BALB/cspleen cell-immunized B10.D2 mice.

The efficiency of MiHL incompatible spleen cells in eradicating residualBCL1 cells was also assayed by adoptive transfer into secondary BALB/crecipients (10 mice in each group) of 10⁵ spleen cells from separatepools of spleen cells made from the animals in each experimental group(at 3 weeks post transplantation) (FIG. 8). Interestingly, 95% ofsecondary BALB/c recipients inoculated with spleen cells obtained fromthe 1st group remained leukemia-free for >120 days. In contrast, only41% of secondary BALB/c recipients inoculated with spleen cells obtainedfrom the 2nd group were alive and leukemia-free for >120 days. Allsecondary BALB/c recipients of cells obtained from groups 3 and 4developed leukemia within 45 (median 38) days. The anti-leukemic effectsinduced with BCL1-immunized B10.D2 spleen cells over both normal,unimmunized B10.D2 spleen cells and BALB/c spleen cell-immunized B10.D2spleen cells were significant (p<0.001 and p=0.027, respectively).Unlike GVL effects induced across an incompatibility involving the MHCand MiHL, BALB/c spleen cell-immunized B10.D2 spleen cells inducedstronger GVL effects compared with normal, unimmunized B10.D2 spleencells (p<0.0001).

FIG. 9 shows that BALB/c mice inoculated with MiHL-incompatible,BCL1-immune B10.D2 spleen cells, after a minor bout of GVHD manifestedby transient weight loss in the first week post grafting, were fullyresistant to GVHD. In contrast, BALB/c mice inoculated with eitherspleen cells from MiHL-incompatible normal, unimmunized B10.D2 mice orspleen cells from MiHL-incompatible BALB/c spleen cell-immunized micedeveloped lethal GVHD. Nevertheless, recipients given BALB/c spleencell-immunized B10.D2 spleen cells survived longer than control animals,indicating that some GVL effects may have been induced but were maskedby GVHD.

EXAMPLE 12 Histological Findings in Mice with Induced GVHD/GVL

As indicated above, mice treated with immunized lymphocytes resistedleukemia development more effectively than recipients of non-immunizedcells, and yet developed less GVHD across MHC and MiHL and MiHL onlybarriers. Of 3 mice that received therapy with normal, unimmunizedspleen cells, 2 had major histological abnormalities in the livercompatible with acute GVHD. Similar liver lesions were demonstrated inonly 1 of 5 mice treated with donor cells from mice immunized with hostspleen cells. None of 4 mice treated with spleen cells fromBCL1-immunized animals had any histological abnormalities in the liver.No leukemic infiltrates were demonstrable in any of the mice treatedacross an incompatibility involving both MHC and MiHL. In micechallenged across MiHL barriers only, injection of normal B10.D2 cellsdid not cause any histological changes consistent with GVHD in theBALB/c recipients, whereas mice injected with B10.D2 spleen cellsobtained from BCL1-immunized or BALB/c spleen cells-immunized B10.D2mice had infiltrations in the lung and in the liver. However, asindicated above, these infiltrations were not associated with increasedGVHD. Leukemia cells were found in the lung and liver of BALB/c miceinjected with normal, unimmunized donor spleen cells and in BALB/crecipients of spleen cells from BALB/c spleen cell-immunized B10.D2mice. In contrast, no leukemic infiltrates were found in mice treatedwith spleen cells obtained from BCL-immunized mice.

EXAMPLE 13 Cytokine Production Correlates with GVHD and GVL Potential

The cytokine profiles of (a) spleen cells from C57BL/6 mice immunizedacross an incompatibility involving both MHC and MiHL alloantigens byinjection of either BCL1 cells or normal BALB/c spleen cells and (b)spleen cells from F1 mice inoculated with BCL1 cells and the spleen cellpopulations recited in (a), were determined.

Results

As shown in FIG. 10, the level of IFN-γ in the supernatant of spleencells of BCL1-immunized C57BL/6 mice was 3 times higher than that ofspleen cells from BALB/c spleen cell-immunized mice and 6 times higherthan that in normal, unimmunized spleen cells. The level of IL-2 in thesupernatant of spleen cells of normal, unimmunized C57BL/6 mice was 4 to5 times higher than the level of IL-2 in the supernatant of spleen cellsobtained from donors immunized with either BCL1 or with BALB/c spleencells (FIG. 11). The level of IL-10 in supernatant of spleen cells fromC57BL/6 mice immunized with BCL1 cells was 2 times higher than that inthe supernatant of spleen cells from C57BL/6 mice immunized with BALB/cspleen cells and of spleen cells from normal, unimmunized C57BL/6 cells(FIG. 12). No differences in the supernatant levels of TNF-α weredetected in spleen cells from normal, unimmunized C57BL/6 mice,BCL1-immunized C57BL/6 mice, and BALB/c spleen cell-immunized C57BL/6mice.

Cytokine levels were also measured in supernatants of cultures of spleencells isolated 3 weeks after cell therapy from F1 recipients of theabove C57BL/6 spleen cells. TNF-α levels in supernatants of spleen cellsfrom F1 mice treated with normal, unimmunized C57BL/6 spleen cells were4-10 times higher than in supernatants of spleen cells from F1 micegiven spleen cells from BCL1 immunized or BALB/c spleen cell-immunizedmice C57BL/6 (FIG. 13). The level of IL-2 in the supernatants of spleencells of F1 mice inoculated with spleen cells from BALB/c spleencell-immunized C57BL/6 mice or with spleen cells from normal,unimmunized C57BL/6 mice were 3 to 4 times higher than in thesupernatant of spleen cells from F1 mice given spleen cells fromBCL1-immunized C57BL/6 mice (FIG. 14). The level of IFN-γ in thesupernatant of spleen cells from F1 mice given spleen cells fromBCL1-immunized C57BL/6 mice was 10 times lower than that in thesupernatant of spleen cells of F1 mice that received normal, unimmunizedC57BL/6 cells (FIG. 15). No differences were detected in the supernatantlevels of IL-10 in spleen cells from F1 mice inoculated with the threetypes of C57BL/6 cells. The level of IL-4 was about five times higher inthe supernatant of spleen cells of F1 recipients of spleen cells fromBCL1-immunized C57BL/6 mice than that in the supernatants of spleencells from F1 mice inoculated with spleen cells from either BALB/cspleen cell-immunized C57BL/6 mice or normal, unimmunized C57BL/6 mice(FIG. 16).

The potent allospecific (MHC and MiHL) tolerizing activity of BCL1 tumorcells, and hence the GVHD suppressive effect of immunization with them,could be due to their presentation of the relevant alloantigens withoutthe participation of co-stimulatory molecules (e.g., B7), a mode ofantigen presentation known to induce tolerance. In addition, thecytokine profiles observed in the experiments described above suggest apossible basis for the seemingly paradoxical findings of (a) enhancedanti-tumor efficacy and (b) concomitant decreased GVH activity inlymphoid cells from donor mice pre-exposed to host tumor cells comparedto lymphoid cells from mice pre-exposed to host hemopoietic (spleen)cells. It is possible that the Th1/Th2 balance, both in the lymphoidcells of the immunized donor animals and in those of the host animals towhich the immunized lymphoid cells are transferred, is differentiallyaffected by the type of immunization given to the donor animal. Thus,for example, production of IL-10 (a prototypical Th2 cytokine) wasup-regulated whereas production of IL-2 (a prototypical Th1 cytokine)was decreased in C57BL/6 spleen cells obtained from mice immunizedacross an MHC barrier with tumor cells compared to spleen cells fromC57BL/6 mice immunized across the same MHC barrier, but with normalspleen cells (FIGS. 11 and 12). In addition, an increased level of IL-4(another prototypical Th2 cytokine) (FIG. 16) accompanied by reducedIL-2 (FIG. 14), TNF-α (FIG. 13) and IFN-γ (FIG. 15) (all prototypicalTh1 cytokines) levels in supernatants of spleen cells from F1 host miceinjected with BCL1-immunized C57BL/6 spleen cells, relative to thelevels in supernatants of spleen cells from F1 host mice injected withspleen cells from BALB/c spleen cell-immunized C57BL/6 donor mice,suggest that the Th2 shifted C57BL/6 donor cells transferred the Th2bias (i.e., higher level of Th2 effects compared to Th1 effects) to theF1 hosts. Furthermore, it is known that Th1-type cytokines are generallyassociated with cellular immune or delayed-type hypersensitivityresponses, while Th2 cytokines are generally associated with humoral(antibody) responses and that GVHD is largely due to cell-mediatedimmunological effects. Thus, in summary, immunization of the donor micewith tumor cells could result in a bias towards Th2 cytokine productionand hence diminished GVHD disease potential in the T cells of the donormice.

It is also possible that, while the Th1 cytokine levels are decreasedsufficiently to minimize GVHD, there may be adequate levels for aneffective cellular anti-tumor (GVL) response. Alternatively, themechanism of the anti-tumor response could differ in some aspects fromthe GVHD response and, as such, could be facilitated by Th2 cytokinesrather than by Th1 cytokines. In addition, not only are Th2 cytokinesgenerally considered not to be “helper” cytokines for cellular immuneresponses, they have been shown to actively suppress cellular immuneresponses. Thus, the Th2 bias transferred to the F1 host mice by cellsfrom BCL1 tumor-immunized donor mice may actually act to activelysuppress GVHD.

The above-described discordancy in GVL and GVHD activity in donor cellsfrom animals immunized with tumor cells versus normal spleen cells couldalso be due to one or a combination of the following effects:

(a) tumor antigen specific effector T cells being relatively resistantto tolerance induction compared to alloantigen (MHC or MiHL) specific Tcells;

(b) the anti-tumor activity being at least partially mediated by non-Tcells (e.g., NK cells) that are activated rather than tolerized by tumorimmunization and/or Th2 cytokines;

(c) tumor cells being relatively more sensitive to allo-specificeffector cells; and

(d) tumor cells being relatively poor inducers of activation inducedapoptosis in allospecific and/or tumor specific effector cells.

While any one or a combination of the above-mechanisms may explain theabove-described dissociation between GVL and GVHD activity, theinvention is not limited by any particular mechanism of action.

EXAMPLE 14 Steps 1-3 of the Tolerization Protocol can be Performed onthe Same Day as Transplantation (“Short Protocol”)

BALB/c recipient mice were administered a single dose of sTLI (200 cGy)and an injection of non T cell depleted BMC from B6 donors on day 0. Onegroup of BALB/c mice treated in this way received B6 BM stromal graftsand a second group received B6 heart grafts. All grafts were alsoperformed on day 0. All mice received an injection of Cy (200 mg/kg) onday 1 and a second injection of B6 BMC on day 2. In light of thesurvival of 100% of the mice in both groups (FIG. 17), GVHD wasprevented by the described protocols. In addition, 100% of the BMstromal grafts and approximately 80% of the heart grafts survived.

This experiment indicated that it is possible to successfully transplantan allograft into a subject at the same time as initiating thetolerogenic method of the invention. It is expected that by simplyaltering doses of, for example, the TLI, BM and/or Cy, as well as thefrequency of subsequent administrations of these agents, it will also bepossible to apply such a “short” protocol to xenogeneic recipient-donorcombinations.

EXAMPLE 15 In Vitro Non-specific Depletion of GVHD Activity

The ability of the drug mafosphamide (ASTA-Z), which is identical to4-hydroxyperoxycyclophosphamide (4HC), to deplete BALB/c mouse spleencells of T cell responsiveness was tested under in vitro conditionsknown to result in preservation of hemopoeitic stem cell activity aftertreatment of hemopoietic cells from humans as well experimental animals(e.g., mice). BALB/c spleen cells were cultured for 30 minutes at 37° C.in tissue culture medium containing 100 μg/ml of ASTA-Z and then testedfor in vitro T cell responses. The exposure to ASTA-Z resulted in theelimination of in vitro proliferative responses to the T cell mitogensconcanavalin A (ConA) and phytohemagglutinin (PHA) (Table 8). Moreover,addition of IL-2 (1,000 IU/ml) to the cultures of spleen cells andASTA-Z did not overcome inhibition of responsiveness to the mitogens bythe ASTA-Z.

TABLE 8 Treatment of murine spleen cells with ASTA-Z results inelimination of responsiveness to T cell mitogens. Cell proliferation^(a)No Treatment Mitogens Con-A PHA Untreated 702 107,277  63,604 ASTA-Z 2192,980  5,118 rIL-2 31,090   52,287  39,390 ASTA-Z + rIL-2 624 4,767 9,453 ^(a)Cell proliferation was measured as counts per minute (cpm) of[³H]-thymidine incorporated into the cells.

On the other hand, ASTA-Z treatment of BALB/c spleen cells (SP) or bonemarrow (BM) cells did not decrease the generation of killer cellscapable of killing murine YAC-1 and P815 tumor target cells by culturingof the SP and BM cells in IL-2 (FIGS. 18 and 19). After treatment of theSP or BM cells with ASTA-Z, as described above, excess ASTA-Z wasremoved and the cells were cultured with human rIL-2 (6,000 IU/ml) for 4days. They were then harvested and tested for cytolytic activity instandard ⁵¹Cr-release assays. While YAC-1 cells are sensitive to lysisby both NK and activated NK cells, P815 cells are not sensitive to lysisby NK cells but are sensitive to activated NK cells. Thus it appearsthat the cytolytic activity detected in the assays was, largely atleast, due to the action of activated NK cells. Furthermore, the sametreatment of human PBMC with ASTA-Z did not decrease the generation ofkiller cells capable of killing human Daudi and K562 tumor target cellsby culturing of the PBMC with human rIL-2 under the same conditionsdescribed above (Table 9). While K562 cells are sensitive to lysis byboth NK and activated NK cells, Daudi cells are not sensitive to NKcells but are sensitive to activated NK cells. Thus, as in the murinesystem described above, the cytotoxic activity was probably due, largelyat least, to NK cells activated by IL-2.

TABLE 9 Treatment of human PBMC with ASTA-Z does not decrease NK cellactivity Lysis of Target Cells^(a) Daudi target cells^(b) K562 targetcells^(b) ASTA-Z ASTA-Z Untreated treated Untreated treated ExperimentPBMC PBMC PBMC PBMC 1 39 71 35 48 2 40 42 44 52 ^(a)Lysis of targetcells was measured as the percentage of ⁵¹Cr released from ⁵¹Cr-labeledtarget cells after incubation with effector cells (at a target cell toeffector cell ratio of 1:100) obtained from cultures containing IL-2(6,000 IU/ml) and either untreated or ASTA-Z treated PBMC. ^(b)K562cells are sensitive to lysis by both activated and unactivated NK cellsand Daudi cells are sensitive to lysis by activated but not unactivatedNK cells.

In parallel murine experiments, culture of B6 bone marrow for 30 minuteswith ASTA-Z (100 μg/ml) prior to injection (25×10⁶ per mouse) intolethally irradiated (1,100 cGy of TBI) SJL/J mice reduced the capacityof the bone marrow cells to induce GVHD (FIG. 20). Similarly, culture ofB6 spleen cells for 30 minutes with ASTA-Z (100 μg/ml) prior toinjection (25×10⁶ per mouse) into sublethally irradiated BALB/c mice(600 cGy) reduced the capacity of the spleen cells to induce GVHD (FIG.21).

Thus, ASTA-Z can be used to deplete hemopoietic cells to be used foreither bone marrow transplantation (e.g., for step 4 of the toleranceprotocol described herein). It can also be used to deplete cells to beused for cell therapy of the allospecific (or xenospecific) T cellreactivity that leads to GVHD while retaining or even being enriched forgraft-versus-tumor (e.g., leukemia) activity which could, at least inpart, be due to the action of NK cells.

EXAMPLE 16 Non-Myeloablative, Donor-Specific Tolerogenic Treatment in aHuman Patient

Patient No. 1 Prior to non-myeloablative conditioning, donor-specifictolerance induction, and allogeneic bone marrow transplantation (ABMT),this male patient underwent autologous stem cell transplantation (ASCT)almost 38 months after diagnosis of Hodgkin's Disease stage III B. Thepatient had failed MOPP/ABVD alternative treatment (8 cycles), radiationtherapy, subsequent treatments with velban, adriamycin, bleomycin andDTIC, and repeated cycles of additional chemotherapy including,following his first overt relapse 2 years after diagnosis, MOPP (4cycles) and VP16, cisplatin, ifosfamide, and uromitexan (5 cycles).Relapse was noted again 2 months after ASCT and the clinical picture offever without obvious infectious etiology suggested persistence of theHodgkin's Disease.

Allogeneic bone marrow transplantation (ABMT), followingnon-myeloablative conditioning and donor-specific tolerization wasoffered to the patient as a possible method of treatment. It wasconsidered that this treatment could overcome long-lasting hypoplasiaand could antagonize the persisting Hodgkin's Disease by inducing graftvs. Hodgkin's Disease tumor cell responses.

Tissue typing data revealed a phenotypic mismatch in HLA class I(serological testing) between the patient and the available donor, hisfather:

Patient: A28 A19 B41 B5

Donor (father): A28 A30 B41 B51

Typing of HLA class II revealed:

Patient: DRB1*1104 DRB1*0404 DQB1*0301 DQB1*0402

Donor (father): DRB1*1101 DRB1*0404 DQB1*0301 DQB1*0402

Starting on day 0, the patient was conditioned non-myeloablatively withFludarabine (30 mg/kg/day) for 3 consecutive days. One day later, thepatient received an infusion of G-CSF mobilized peripheral blood cells(“first allograft”) collected form his father (2.98×10⁸ nucleatedcells/kg) as a source of donor-specific antigens, followed by 3 dailynon-myeloablative period doses of cytoxan 60 mg/kg (4,500 mg daily) toeliminate donor-specific alloreactive T cells. An infusion of unselectedpaternal bone marrow cells (9.6×10⁸ nucleated cells/kg) was carried out(“second allograft”) one day after termination of the last dose ofcytoxan. It was decided to use unmodified bone marrow cells with nofurther T cell depletion for the second allograft in order to maximizethe chance of stem cell engraftment on the one hand as well as GVTeffects on the other.

Fewer up to almost 40° C. developed in the first week after the secondallograft and the patient required frequent single donor plateletinfusions for prevention of bleeding. The patient also receivedantibiotic therapy with amikacin, tazocin, preventive therapy againstfungal infection with diflucan and acyclovir therapy againstcytomegalovirus infection. Since fever did not respond completely toantibiotic therapy, amphotericin B (1 mg/kg) was given every other day.Fever persisted throughout hospitalization. The patient's white bloodcell count (WBC) rose to 1.0×10⁹/L on day +14 and his absoluteneutrophil count (ANC) reached ≧0.5×10⁹/L on day +14 and ≧1.0×10⁹/L onday +28. The WBC rose gradually to a maximal level of 5.1×10⁹/L with 75%granulocytes. However, thrombocytopenia persisted. Engraftment wasconfirmed by rising counts and by detection of donor DNA by the VariableNumber of Tandem Repeats-Polymerase Chain Reaction (VNTR-PCR), atechnique known to those in the art.

On day +10, the patient experienced a grand mal seizure which respondedto valium infusion. No focal neurologic findings were found except thatthe Babinski's sign was positive bilaterally. Cyclosporine A wasadministered as a prophylactic treatment for GVHD. Overt skin rashtypical of GVHD appeared on day +12. Liver manifestations developedsubsequently. Despite combination therapy with solumedrol (2 mg/kg)daily and cyclosporine, with continuation of the antibiotic andanti-fungal therapy, the patient's condition deteriorated gradually,with diarrhea up to 12 times a day, starting on day +16, a symptomindicative of stage IV GVHD. Despite intensive treatment of both GVHDand potential infections, spikes of fever continued with dyspnea thatdeveloped in parallel with pulmonary bleeding and bilateral interstitialinfiltration in the lungs on day +28. The patient was intubated on day+29. Large volumes of secretion were aspirated through the tube. Thesecretions included blood but lavage did not reveal any infectiousagent. Despite intensive therapy including dopamine drip and carefulmaintenance of pulmonary system, the blood pressure dropped graduallyand the patient expired on day +29.

In conclusion, the successful engraftment of the patient by his father'sbone marrow used for the second allograft indicated that HLA mismatchedstem cells can be accepted following selective depletion of host cellswith the capacity to reject donor alloantigenic tissue and withoutmyeloablative conditioning. Developments in the patient suggest that,due to pancytopenia following ASCT and the failure to establish a highlevel of protective mixed chimerism, he may have been more susceptibleto GVHD. Non T cell depleted bone marrow was used for the secondallograft and this unfortunately resulted in GVHD. Nevertheless, theabove-described findings demonstrated that HLA mismatched cells can beaccepted and engrafted without myeloablative conditioning using thedescribed tolerogenic protocol.

These data considered in light of murine experiments indicate that itwill be possible to obtain engraftment in human patients without GVHD ifthe donor bone marrow used for the second is allograft is either (a)depleted of T-cells prior to infusion or (b) is used undepleted but atransient stage of mixed chimerism in the recipient is achieved.Furthermore, the combined findings of this clinical study and the murineexperiments indicate that, in human patients, it will be possible toprevent rejection of allografts if the recipient is depleted ofdonor-specific T cells prior to the allograft.

EXAMPLE 17 Effective Treatment of Human Chronic Myelogenous Leukemia(CML) with Allogeneic Lymphocytes Pre-exposed to Alloantigens of thePatient

Allogeneic cell therapy using donor lymphocytes pre-exposed toalloantigens of the patient was given to a female patient withPhiladelphia chromosome-positive (Ph+) CML who had relapsed 9 monthsafter allogeneic bone marrow transplantation. The bone marrow cells usedfor the transplant were from a HLA-A, -B, -C and -DR identical 6-monthold brother (the “donor”). The patient had failed to respond to severalrounds of allogeneic cell therapy (given subsequent to the bone marrowtransplant) using donor PBMC that, in some of the treatments, wereactivated with IL-2. The patient's bone marrow contained approximately95% Ph+ cells prior to this allogeneic cell therapy which consisted ofthe following sequential procedures.

(a) 10⁷ donor PBMC per kg were administered i.v. 24 hours after a lowdose of Cy (500 mg/m²). No remission was obtained.

(b) 10⁷ donor PBMC (activated in vitro with IL-2) per kg wereadministered i.v.. Beginning on the day of cell infusion, rIL-2 (6×10⁶IU/m²/day) was administered subcutaneously for 3 days. The wholeprocedure was performed twice, approximately one month apart, andresulted in a transient decrease to about 67% in the proportion of Ph+cells in the patient's bone marrow.

(c) 6×10⁶ paternal PBMC (activated in vitro with IL-2) were administeredi.v., resulting in a transient decrease to about 60% in the proportionof Ph+ cells in the patient's bone marrow. This decrease was followed bya gradual increase to 94% Ph+ bone marrow cells.

At this time it was decided to treat the patient with donor PBMCactivated in vitro against alloantigens of the patient. The patient wasinfused with donor PBMC that had been exposed twice in vitro for 1 dayto irradiated (3,000 cGy) PBMC from both parents of the patient (anddonor). The donor PBMC were thus activated to parental MHC antigens notexpressed by the patient and MiHL antigens expressed by the patient butnot the donor, the patient and donor being HLA identical children of theparents. The patient was then given rIL-2 (6×10⁶ IU/m²/day) for 3 days,starting on the day of cell infusion. The whole procedure was carriedout twice, approximately one month apart. Interferon-α (1.5×10⁶) wasadministered 3 times a week for 4 years. The patient has now been inremission for greater than five years. She has no detectable leukemiacells (both by karyotype analysis and RT-PCR to detect mRNA transcriptsderived from a bcr/abl hybrid DNA sequence produced by the Philadelphiat(9;22)(q34;q11) chromosomal translocation), 100% of both her blood andher bone marrow cells are donor-derived, and she has no clinical signsof GVHD.

This study indicates that the efficacy of allogeneic cell therapy ofhuman cancer can be enhanced by pre-exposure of the donor cells to beused for therapy to alloantigens expressed by the patient. In light ofthe above-described experiments in mice, such pre-exposed donor cellsare also likely to display decreased GVH activity compared to unexposedcells.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of inducing allogeneic donor-specifictolerance in a host mammal to a graft from an allogeneic donor mammalcomprising: (a) administering a short course of total lymphoidirradiation (sTLI) to said host mammal; (b) administering allogeneicdonor antigens selected from a group consisting of donor blood cells anddonor bone marrow cells to said host mammal; (c) administering animmunosuppressive agent to said host mammal in a non-myeloablativeregimen sufficient to decrease said host mammal's functional Tlymphocyte population; (d) transplanting cells, a tissue, or an organfrom said donor into said host mammal; (e) administering anon-myeloablative dose of cyclophosphamide to said host mammal toselectively eliminate said host mammal's lymphocytes responding to saiddonor antigens, wherein said cyclophosphamide is administered for 1-2days; (f) administering allogeneic donor antigens derived from the samedonor mammal as in step (b) selected from a group consisting of donorblood cells and donor bone marrow cells to said host mammal; whereinsteps (a)-(d) are performed on the same day.
 2. The method of claim 1,wherein step (f) is performed prior to step (d).
 3. The method of claim1, wherein steps (a)-(d) are performed prior to steps (e) and (f).
 4. Amethod of inducing concordant xenogeneic donor-specific tolerance in ahost mammal to a graft from a concordant xenogeneic donor mammalcomprising: (a) administering a short course of total lymphoidirradiation (sTLI) to said host mammal; (b) administering concordantxenogeneic donor antigens selected from a group consisting of donorblood cells and donor bone marrow cells to said host mammal; (c)administering an immunosuppressive agent to said host mammal in anon-myeloablative regimen sufficient to decrease said host mammal'sfunctional T lymphocyte population; (d) transplanting cells, a tissue,or an organ from said concordant xenogeneic donor into said host mammal;(e) administering a non-myeloablative dose of cyclophosphamide to saidhost mammal to selectively eliminate said host mammal's lymphocytesresponding to said donor antigens, wherein said cyclophosphamide isadministered for 1-2 days; (f) administering allogeneic donor antigensderived from the same donor mammal as in step (b) selected from a groupconsisting of donor blood cells and donor bone marrow cells to said hostmammal; wherein steps (a)-(d) are performed on the same day.
 5. Themethod of claim 4, wherein step (f) is performed prior to step (d). 6.The method of claim 4, wherein steps (a)-(d) are performed prior tosteps (e) and (f).
 7. A method of inducing discordant xenogeneicdonor-specific tolerance in a host mammal to a graft from a discordantxenogeneic donor mammal comprising: (a) removal of naturally occurringhost anti-xenogeneic antibodies (b) administering a short course oftotal lymphoid irradiation (sTLI) to said host mammal; (c) administeringdiscordant xenogeneic donor antigens selected from a group consisting ofdonor blood cells and donor bone marrow cells to said host mammal; (d)administering an immunosuppressive agent to said host mammal in anon-myeloablative regimen sufficient to decrease said host mammal'sfunctional T lymphocyte population; (e) transplanting cells, a tissue,or an organ from said discordant xenogeneic donor into said host mammal;(f) administering a non-myeloablative dose of cyclophosphamide to saidhost mammal to selectively eliminate said host mammal's lymphocytesresponding to said donor antigens, wherein said cyclophosphamide isadministered for 1-2 days; (g) administering allogeneic donor antigensderived from the same donor mammal as in step (b) selected from a groupconsisting of donor blood cells and donor bone marrow cells to said hostmammal; wherein steps (a)-(e) are performed on the same day.
 8. Themethod of claim 7, wherein step (g) is performed prior to step (e). 9.The method of claim 7, wherein steps (a)-(e) are performed prior tosteps (f) and (g).